Human virus causing respiratory tract infection and uses thereof

ABSTRACT

The present invention provides the complete genomic sequence of a novel human coronavirus, coined as coronavirus-HKU1 (“CoV-HKU1”), isolated in Hong Kong from a patient who had a recent history of visit to Schenzhen, China. The virus belongs to the order Nidovirales of the family Coronaviridae, being a single-stranded RNA virus of positive polarity. The invention also provides the deduced amino acid sequences of the complete genome of the CoV-HKU1. The nucleotide sequences and deduced amino acid sequences of the CoV-HKU1 are useful in preventing, diagnosing and/or treating the infection by CoV-HKU1. Furthermore, the invention provides immunogenic and vaccine preparations using recombinant and chimeric forms as well as subunits of the CoV-HKU1 based on the nucleotide sequences and deduced amino acid sequences of the CoV-HKU1.

SEQUENCE LISTING

The instant application contains a “lengthy” Sequence Listing which hasbeen submitted via CD-R in lieu of a printed paper copy, and is herebyincorporated by reference in its entirety. Said CD-R, recorded on Jul.13, 2004, are labeled “Copy 1” and “Copy 2”, respectively, and eachcontains only one identical 1.54 Mb file (V6900031.APP).

1. INTRODUCTION

The present invention relates to a novel virus causing respiratory tractinfection in humans [“coronavirus-HKU1 (CoV-HKU1)”]. The CoV-HKU1 isidentified to be phylogenetically similar to known members ofCoronaviridae. The present invention relates to a nucleotide sequencecomprising the complete genomic sequence of the CoV-HKU1. The inventionfurther relates to nucleotide sequences comprising a portion of thegenomic sequence of the CoV-HKU1. The invention also relates to thededuced amino acid sequences of the complete genome of the CoV-HKU1. Theinvention further relates to the nucleic acids and peptides encoded byand/or derived from these sequences and their use in diagnostic methodsand therapeutic methods, such as for immunogens. The invention furtherencompasses chimeric or recombinant viruses encoded by said nucleotidesequences and antibodies directed against polypeptides encoded by thenucleotide sequence. Furthermore, the invention relates to vaccinepreparations comprising the CoV-HKU1 recombinant and chimeric forms ofsaid virus as well as protein extracts and subunits of said virus.

2. BACKGROUND OF THE INVENTION

In January, 2004, a 71-year-old Chinese man was admitted to hospitalbecause of fever and chills for two days associated with sore throat,rhinorrhoea, productive cough with purulent sputum, headache and nausea.He had history of pulmonary tuberculosis more than 40 years agocomplicated by cicatrization of right upper lobe and bronchiectasis withchronic Pseudomonas aeruginosa colonization of airways. He was a chronicsmoker and also had chronic obstructive airway disease, hyperlipidemia,and asymptomatic abdominal aortic aneurysm. He had just returned fromShenzhen of China three days before admission. During his three-day tripto Shenzhen, he had no history of contact with or consumption of wildanimals. On admission, his oral temperature was 37.6° C. Physicalexamination showed tracheal deviation to the right and inspiratorycrackles over the anterior left lower zone. His haemoglobin level was14.7 g/dL, total white cell count 12.1×10⁹/L, with neutrophil 9.7×10⁹/L,lymphocyte 1.6×10⁹/L and monocyte 0.5×10⁹/L, and plate count 303×10⁹/L.His liver and renal function tests were within normal limits. Chestradiograph showed right upper lobe collapse and new patchy infiltratesover the left lower zone. Blood culture was performed. Empirical oralamoxicillin/clavulanate and azithromycin were commenced. Nasopharyngealaspirates for direct antigen detection for respiratory viruses, RT-PCRfor influenza A virus, human metapneumovirus and SARS-CoV, and viralcultures were negative. Sputum for bacterial culture only recovered P.aeruginosa. Sputum for mycobacterial culture was negative. Blood culturewas negative. Paired sera for antibodies against Mycoplasma, Chlamydia,Legionella, and SARS-CoV did not show any rise in antibody titres. Hisfever subsided two days after admission. His cough improved and he wasdischarged after five days of hospitalization. Amoxicillin/clavulanateand azithromycin were continued for a total of seven days. The presentinventors were the group involved in the investigation of this patient.All tests for identifying commonly recognized viruses and bacteria werenegative in these patients. The etiologic agent responsible for thisdisease was not known until the complete genome of CoV-HKU1 from thispatient by the present inventors as disclosed herein. Namely, thepresent invention discloses a novel human virus that has been identifiedfrom a patient suffering from pneumonia. The invention is useful in bothclinical and scientific research applications.

3. SUMMARY OF INVENTION

The present invention is based upon the inventor's complete genomesequencing of a novel virus (“CoV-HKU1”) causing pneumonia in humans.The virus was discovered from a patient suffering from pneumonia in HongKong. The virus is a single-stranded RNA virus of positive polaritywhich belongs to the order, Nidovirales, of the family, Coronaviridae.Accordingly, the invention relates to CoV-HKU1 that phylogeneticallyrelates to known members of Coronaviridae. In a specific embodiment, theinvention provides complete genomic sequence of CoV-HKU1. In a preferredembodiment, the virus comprises a nucleotide sequence of SEQ ID NO:1and/or 3. In another specific embodiment, the invention provides nucleicacids isolated from the virus. The virus preferably comprises anucleotide sequence of SEQ ID NO:1 and/or 3 in its genome. In a specificembodiment, the present invention provides isolated nucleic acidmolecules comprising or, alternatively, consisting of the nucleotidesequence of SEQ ID NO:1, a complement thereof or a portion thereof,preferably at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 100, 150, 200,300, 350, or more contiguous nucleotides of the nucleotide sequence ofSEQ ID NO:1, or a complement thereof. In another specific embodiment,the present invention provides isolated nucleic acid moleculescomprising or, alternatively, consisting of the nucleotide sequence ofSEQ ID NO:3, a complement thereof or a portion thereof, preferably atleast 5, 10, 15, 20, 25, 30, 35, 40, 45, 100, 150, 200, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,050,1,100, 1,150, 1,200, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000,9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000,18,000, 19,000, 20,000, 21,000, 22,000, 23,000, 24,000, 25,000, 26,000,27,000, 28,000, 29,000 or more contiguous nucleotides of the nucleotidesequence of SEQ ID NO:3, or a complement thereof. Furthermore, inanother specific embodiment, the invention provides isolated nucleicacid molecules which hybridize under stringent conditions, as definedherein, to a nucleic acid molecule having the sequence of SEQ ID NO:1 or3, or a complement thereof. In preferred embodiments, such nucleic acidmolecules encode amino acid sequences that have biological activitiesexhibited by the polypeptides encoded by the nucleotide sequence of SEQID NO:1 or 3. In another specific embodiment, the invention providesisolated polypeptides or proteins that are encoded by a nucleic acidmolecule comprising or, alternatively consisting of a nucleotidesequence that is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 100, 150,200, 300, 350, or more contiguous nucleotides of the nucleotide sequenceof SEQ ID NO:1, or a complement thereof. In yet another specificembodiment, the invention provides isolated polypeptides or proteinsthat are encoded by a nucleic acid molecule comprising or, alternativelyconsisting of a nucleotide sequence that is at least 5, 10, 15, 20, 25,30, 35, 40, 45, 100, 150, 200, 300, 350, 400, 450, 500, 550, 600, 650,700, 750, 800, 850, 900, 950, 1,000, 1,050, 1,100, 1,150, 1,200, 2,000,3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000,13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 21,000,22,000, 23,000, 24,000, 25,000, 26,000, 27,000, 28,000, 29,000 or morecontiguous nucleotides of the nucleotide sequence of SEQ ID NO:3, or acomplement thereof. The polypeptides or proteins include those havingthe amino acid sequences of SEQ ID NO:2 and SEQ ID NOS:34-2918 shown inFIGS. 2 and 3, respectively. The invention further provides proteins orpolypeptides that are isolated from the CoV-HKU1, including viralproteins isolated from cells infected with the virus but not present incomparable uninfected cells. The polypeptides or the proteins of thepresent invention preferably have a biological activity of the protein(including antigenicity and/or immunogenicity) encoded by the nucleotidesequence that is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 100, 150,200, 300, 350, or more contiguous nucleotides of the nucleotide sequenceof SEQ ID NO:1. In other embodiments, the polypeptides or the proteinsof the present invention have a biological activity of the protein(including antigenicity and/or immunogenicity) encoded by a nucleotidesequence that is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 100, 150,200, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950, 1,000, 1,050, 1,100, 1,150, 1,200, 2,000, 3,000, 4,000, 5,000,6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000,15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 21,000, 22,000, 23,000,24,000, 25,000, 26,000, 27,000, 28,000, 29,000 or more contiguousnucleotides of the nucleotide sequence of SEQ ID NO:3, or a complementthereof.

In one aspect, the invention relates to the use of CoV-HKU1 fordiagnostic methods. In a specific embodiment, the invention provides amethod of detecting in a biological sample an antibody thatimmunospecifically binds to the CoV-HKU1, or any proteins orpolypeptides thereof. In another specific embodiment, the inventionprovides a method of detecting in a biological sample an antibody thatimmunospecifically binds to the CoV-HKU1-infected cells. In yet anotherspecific embodiment, the invention provides a method of screening for anantibody that immunospecifically binds and neutralizes CoV-HKU1. Such anantibody is useful for a passive immunization or immunotherapy of asubject infected with CoV-HKU1.

The invention further relates to the use of the sequence information ofthe isolated virus for diagnostic methods. In a specific embodiment, theinvention provides nucleic acid molecules which are suitable for use asprimers consisting of or comprising the nucleotide sequence of SEQ IDNO:1 or 3, a complement thereof, or at least a portion of the nucleotidesequence thereof. In another specific embodiment, the invention providesnucleic acid molecules which are suitable for hybridization to CoV-HKU1nucleic acid, including, but not limited to, as PCR primers, ReverseTranscriptase primers, probes for Southern or Northern analysis or othernucleic acid hybridization analysis for the detection of CoV-HKU1nucleic acids, e.g., consisting of or comprising the nucleotide sequenceof SEQ ID NO:1 or 3, a complement thereof, or a portion thereof.

The invention further provides antibodies that specifically bind apolypeptide of the invention encoded by the nucleotide sequence of SEQID NO:1 or 3 or a fragment thereof, including the polypeptide having theamino acid sequence of SEQ ID NO:2 or SEQ ID NOS:34-2918 shown in FIGS.2 and 3, or encoded by a nucleic acid comprising a nucleotide sequencethat hybridizes under stringent conditions to the nucleotide sequence ofSEQ ID NO:1 or 3 and/or any CoV-HKU1 epitope, having one or morebiological activities of a polypeptide of the invention. The inventionfurther provides antibodies that specifically bind cells or tissues thatare infected by CoV-HKU1. Such antibodies include, but are not limitedto polyclonal, monoclonal, bi-specific, multi-specific, human,humanized, chimeric antibodies, single chain antibodies, Fab fragments,F(ab′)₂ fragments, disulfide-linked Fvs, intrabodies and fragmentscontaining either a VL or VH domain or even a complementary determiningregion (CDR) that specifically binds to a polypeptide of the invention.

In one embodiment, the invention provides methods for detecting thepresence, activity or expression of the CoV-HKU1 of the invention in abiological material, such as cells, blood, saliva, urine, and so forth.The increased or decreased activity or expression of the CoV-HKU1 in asample relative to a control sample can be determined by contacting thebiological material with an agent which can detect directly orindirectly the presence, activity or expression of the CoV-HKU1. In aspecific embodiment, the detecting agents are the antibodies or nucleicacid molecules of the present invention. Antibodies of the invention mayalso be used to detect and/or treat other coronaviruses, such as SevereAcute Respiratory Syndrome (“SARS”) viruses.

In another embodiment, the invention provides vaccine preparations,comprising the CoV-HKU1 recombinant and chimeric forms of said virus, orprotein subunits of the virus. In a specific embodiment, the presentinvention provides methods of preparing recombinant or chimeric forms ofCoV-HKU1. In another specific invention, the vaccine preparations of thepresent invention comprise a nucleic acid or fragment of the CoV-HKU1,or nucleic acid molecules having the sequence of SEQ ID NO:1 or 3, or afragment thereof. In another embodiment, the invention provides vaccinepreparations comprising one or more polypeptides isolated from orproduced from nucleic acid of CoV-HKU1. In a specific embodiment, thevaccine preparations comprise a polypeptide of the invention encoded bythe nucleotide sequence of SEQ ID NO:1 or 3, or a fragment thereof,including the polypeptides having the amino acid sequences of SEQ IDNO:2 or SEQ ID NOS:34-2918 shown in FIGS. 2 and 3, respectively.Furthermore, the present invention provides methods for treating,ameliorating, managing or preventing respiratory tract infections causedby CoV-HKU1 by administering to a subject in need thereof the anti-viralagents of the present invention, alone or in combination with variousanti-viral agents as well as adjuvants, and/or other pharmaceuticallyacceptable excipients.

In another aspect, the present invention provides methods for preventingor inhibiting, under a physiological condition, binding to a host cell,or infection of a host cell, or replication in a host cell, of CoV-HKU1or a virus comprising a nucleic acid molecule comprising the nucleotidesequence of SEQ ID NO:1 or 3 or a complement thereof, by administeringto the host cell the anti-viral agents of the present invention, aloneor in combination with other anti-viral agents. In a specificembodiment, the anti-viral agent of the invention includes theimmunogenic preparations of the invention or an antibody thatimmunospecifically binds CoV-HKU1 or any CoV-HKU1 epitope and/orneutralizes CoV-HKU1. In another specific embodiment, the anti-viralagent is a polypeptide or protein of the present invention or a nucleicacid molecule of the invention. In a specific embodiment, the host cellis a mammalian cell, including a cell of human, primates, cows, horses,sheep, pigs, fowl (e.g., chickens), goats, cats, dogs, hamsters, miceand rats. Preferably a host cell is a primate cell, and most preferablya human cell. Furthermore, the present invention provides pharmaceuticalcompositions comprising anti-viral agents of the present invention and apharmaceutically acceptable carrier. The invention also provides kitscontaining a pharmaceutical composition of the present invention.

3.1 Definitions

The term “an antibody or an antibody fragment that immunospecificallybinds a polypeptide of the invention” as used herein refers to anantibody or a fragment thereof that immunospecifically binds to thepolypeptide encoded by the nucleotide sequence of SEQ ID NO:1 or 3, or afragment thereof, and does not non-specifically bind to otherpolypeptides. An antibody or a fragment thereof that immunospecificallybinds to the polypeptide of the invention may cross-react with otherantigens. Preferably, an antibody or a fragment thereof thatimmunospecifically binds to a polypeptide of the invention does notcross-react with other antigens. An antibody or a fragment thereof thatimmunospecifically binds to the polypeptide of the invention, can beidentified by, for example, immunoassays or other techniques known tothose skilled in the art.

An “isolated” or “purified” peptide or protein is substantially free ofcellular material or other contaminating proteins from the cell ortissue source from which the protein is derived, or substantially freeof chemical precursors or other chemicals when chemically synthesized.The language “substantially free of cellular material” includespreparations of a polypeptide/protein in which the polypeptide/proteinis separated from cellular components of the cells from which it isisolated or recombinantly produced. Thus, a polypeptide/protein that issubstantially free of cellular material includes preparations of thepolypeptide/protein having less than about 30%, 20%, 10%, 5%, 2.5%, or1%, (by dry weight) of contaminating protein. When thepolypeptide/protein is recombinantly produced, it is also preferablysubstantially free of culture medium, i.e., culture medium representsless than about 20%, 10%, or 5% of the volume of the proteinpreparation. When polypeptide/protein is produced by chemical synthesis,it is preferably substantially free of chemical precursors or otherchemicals, i.e., it is separated from chemical precursors or otherchemicals which are involved in the synthesis of the protein.Accordingly, such preparations of the polypeptide/protein have less thanabout 30%, 20%, 10%, 5% (by dry weight) of chemical precursors orcompounds other than polypeptide/protein fragment of interest. In apreferred embodiment of the present invention, polypeptides/proteins areisolated or purified.

An “isolated” nucleic acid molecule is one which is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid molecule. Moreover, an “isolated” nucleic acid molecule,such as a cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. In a preferred embodiment of the invention,nucleic acid molecules encoding polypeptides/proteins of the inventionare isolated or purified. The term “isolated” nucleic acid molecule doesnot include a nucleic acid that is a member of a library that has notbeen purified away from other library clones containing other nucleicacid molecules.

The term “portion” or “fragment” as used herein refers to a fragment ofa nucleic acid molecule containing at least about 10, 15, 25, 30, 35,40, 45, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350,2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000,12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000,21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000, 28,000, 29,000,or more contiguous nucleic acids in length of the relevant nucleic acidmolecule and having at least one functional feature of the nucleic acidmolecule (or the encoded protein has one functional feature of theprotein encoded by the nucleic acid molecule); or a fragment of aprotein or a polypeptide containing at least 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 120, 140, 160, 180, 200,220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 500, 600, 700, 800,900, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,100, 4,200,4,300, 4,350, 4,360, 4,370, 4,380 amino acid residues in length of therelevant protein or polypeptide and having at least one functionalfeature of the protein or polypeptide.

The term “having a biological activity of the protein” or “havingbiological activities of the polypeptides of the invention” refers tothe characteristics of the polypeptides or proteins having a commonbiological activity similar or identical structural domain and/or havingsufficient amino acid identity to the polypeptide encoded by thenucleotide sequence of SEQ ID NO:1 or 3, or the polypeptide having theamino acid sequence of SEQ ID NO:2, or a complement thereof. Such commonbiological activities of the polypeptides of the invention includeantigenicity and immunogenicity.

The term “under stringent condition” refers to hybridization and washingconditions under which nucleotide sequences having at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95%identity to each other remain hybridized to each other. Suchhybridization conditions are described in, for example but not limitedto, Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1-6.3.6.; Basic Methods in Molecular Biology, ElsevierScience Publishing Co., Inc., N.Y. (1986), pp. 75-78, and 84-87; andMolecular Cloning, Cold Spring Harbor Laboratory, N.Y. (1982), pp.387-389, and are well known to those skilled in the art. A preferred,non-limiting example of stringent hybridization conditions ishybridization in 6× sodium chloride/sodium citrate (SSC), 0.5% SDS atabout 68° C. followed by one or more washes (e.g., about 5 to 30 mineach) in 2×SSC, 0.5% SDS at room temperature. Another preferred,non-limiting example of stringent hybridization conditions ishybridization in 6×SSC at about 45° C. followed by one or more washes(e.g., about 5 to 30 min each) in 0.2×SSC, 0.1% SDS at about 45-65° C.

The term “variant” as used herein refers either to a naturally occurringgenetic mutant of CoV-HKU1 or a recombinantly prepared variation ofCoV-HKU1 each of which contain one or more mutations in its genomecompared to CoV-HKU1. The term “variant” may also refers either to anaturally occurring variation of a given peptide or a recombinantlyprepared variation of a given peptide or protein in which one or moreamino acid residues have been modified by amino acid substitution,addition, or deletion.

4. DESCRIPTION OF FIGURES

FIG. 1 shows a partial DNA sequence (SEQ ID NO:1) and its deduced aminoacid sequence (SEQ ID NO:2) obtained from CoV-HKU1 that has 91% aminoacid identity to the RNA-dependent RNA polymerase protein of knownCoronaviruses.

FIG. 2 shows the entire genomic DNA sequence (SEQ ID NO:3) of CoV-HKU1and its deduced amino acid sequences therefrom in three frames. Anasterisk (*) indicates a stop codon which marks the end of a peptide.The first-frame translation and amino acid sequences: SEQ ID NOS:34-456;the second-frame translation and amino acid sequences: SEQ IDNOS:457-723; and the third-frame translation and amino acid sequences:SEQ ID NOS:724-1318.

FIG. 3 shows the complement (SEQ ID NO:1319) of the entire genomic DNAsequence (SEQ ID NO:3) of CoV-HKU1 in 3′→5′ orientation and its deducedamino acid sequences therefrom in three frames. An asterisk (*)indicates a stop codon which marks the end of a peptide. The first-frametranslation and amino acid sequences: SEQ ID NOS:1319-1907; thesecond-frame translation and amino acid sequences: SEQ ID NO:1908-2453;and the third-frame translation and amino acid sequences: SEQ IDNOS:2454-2918.

FIG. 4 shows the genome organization of CoV-HKU1. Arrows indicate theputative cleavage sites of the polyprotein encoded by ORF 1a and ORF 1b.The peptides are shown in SEQ ID NOS:15-17, respectively, in order ofappearance.

FIG. 5A shows the phylogenetic analysis of the chymotrypsin likeprotease (3CL^(pro)), replicase (Rep), helicase (Hel), and hemagglutininesterase (HE); and FIG. 5B shows that of the spike (S), envelope (E),membrane (M), and nucleocapsid (N) proteins of CoV-HKU1. The trees wereconstructed by the neighbor joining method using the Jukes-Cantorcorrection and bootstrap values were calculated from 1000 trees. A totalof 303, 928, 603, 386, 1356, 82, 223 and 441 amino acid positions in3CL^(pro), Rep, Hel, HE, S, E, M, and N respectively were included inthe analysis. The scale bar indicates the estimated number ofsubstitutions per 10 amino acids.

FIG. 6 shows the important features of the S protein of CoV-HKU1(residues 7-336 of SEQ ID NO:420) in comparison with those of otherviruses, i.e., HCoV-OC43 (human coronavirus OC43; SEQ ID NO:21), MHV(murine hepatitis virus; SEQ ID NO:22), SDAV (rat sialodacryoadenitisencephalomyelitis virus; SEQ ID NO:23), BCoV (bovine coronavirus; SEQ IDNO:24), PHEV (porcine hemagglutinating encephalomyelitis virus; SEQ IDNO:25), and ECoV (equine coronavirus; SEQ ID NO:26). The cleavage sitepeptides are shown in residues 752-766 of SEQ ID NO:420 and SEQ IDNOS:28-33, respectively, in order of appearance.

FIG. 7 shows the sequential quantitative RT-PCR (closed squares;copies/ml) for CoV-HKU1 in nasopharyngeal aspirates; and serum IgGantibody titers against N protein of CoV-HKU1 (closed triangles).

FIG. 8 shows the Western blot analysis of purified recombinant CoV-HKU1N protein antigen. Prominent immunoreactive protein bands of about 53kDa were detected by the Western blot using the patient's sera obtainedduring the second and fourth weeks of the illness (lanes 2 and 3). Onlyvery faint bands were observed with the serum samples obtained from thepatient during the first week of the illness (lane 1) and two healthyblood donors (lane 4 and 5), respectively.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the CoV-HKU1 that phylogeneticallyrelates to known Coronaviruses. In a specific embodiment, CoV-HKU1comprises a nucleotide sequence of SEQ ID NO:1 and/or 3. In a specificembodiment, the present invention provides isolated nucleic acidmolecules of the CoV-HKU1, comprising, or, alternatively, consisting ofthe nucleotide sequence of SEQ ID NO:1 and/or 3, a complement thereof ora portion thereof. In another specific embodiment, the inventionprovides isolated nucleic acid molecules which hybridize under stringentconditions, as defined herein, to a nucleic acid molecule having thesequence of SEQ ID NO:1 or 3, or specific genes of known member ofCoronaviridae, or a complement thereof. In another specific embodiment,the invention provides isolated polypeptides or proteins that areencoded by a nucleic acid molecule comprising a nucleotide sequence thatis at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 100, 150, 200, 300,350, or more contiguous nucleotides of the nucleotide sequence of SEQ IDNO:1, or a complement thereof. In yet another specific embodiment, theinvention provides isolated polypeptides or proteins that are encoded bya nucleic acid molecule comprising or, alternatively consisting of anucleotide sequence that is at least 5, 10, 15, 20, 25, 30, 35, 40, 45,100, 150, 200, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,850, 900, 950, 1,000, 1,050, 1,100, 1,150, 1,200, 2,000, 3,000, 4,000,5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000,14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 21,000, 22,000,23,000, 24,000, 25,000, 26,000, 27,000, 28,000, 29,000 or morecontiguous nucleotides of the nucleotide sequence of SEQ ID NO:3, or acomplement thereof. The polypeptides or the proteins of the presentinvention preferably have one or more biological activities of theproteins encoded by the sequence of SEQ ID NO:1, 3, or the native viralproteins containing the amino acid sequences encoded by the sequence ofSEQ ID NO:1 or 3.

The invention further relates to the use of the sequence information ofthe isolated virus for diagnostic and therapeutic methods. In a specificembodiment, the invention provides the entire nucleotide sequence ofCoV-HKU1 (SEQ ID NO:3), or fragments, or complement thereof.Furthermore, the present invention relates to a nucleic acid moleculethat hybridizes any portion of the genome of the CoV-HKU1 (SEQ ID NO:3)under the stringent conditions. In a specific embodiment, the inventionprovides nucleic acid molecules which are suitable for use as primersconsisting of or comprising the nucleotide sequence of SEQ ID NO:1 or 3,or a complement thereof, or a portion thereof. In another specificembodiment, the invention provides nucleic acid molecules which aresuitable for use as hybridization probes for the detection of nucleicacids encoding a polypeptide of the invention, consisting of orcomprising the nucleotide sequence of SEQ ID NO:1 or 3, a complementthereof, or a portion thereof. The invention further encompasseschimeric or recombinant viruses or viral proteins encoded by saidnucleotide sequences.

The invention further provides antibodies that specifically bind apolypeptide of the invention encoded by the nucleotide sequence of SEQID NO:1 or 3, or a fragment thereof, or any CoV-HKU1 epitope as well asthe polypeptides having the amino acid sequences of SEQ ID NO:2 and SEQID NOS:34-2918, respectively, shown in FIGS. 2 and 3. Such antibodiesinclude, but are not limited to polyclonal, monoclonal, bi-specific,multi-specific, human, humanized, chimeric antibodies, single chainantibodies, Fab fragments, F(ab′)₂ fragments, disulfide-linked Fvs,intrabodies and fragments containing either a VL or VH domain or even acomplementary determining region (CDR) that specifically binds to apolypeptide of the invention.

In one embodiment, the invention provides methods for detecting thepresence, activity or expression of the CoV-HKU1 of the invention in abiological material, such as cells, blood, saliva, urine, sputum,nasopharyngeal aspirates, and so forth. The presence of the CoV-HKU1 ina sample can be determined by contacting the biological material with anagent which can detect directly or indirectly the presence, activity orexpression of the CoV-HKU1. In a specific embodiment, the detectionagents are the antibodies of the present invention. In anotherembodiment, the detection agent is a nucleic acid of the presentinvention.

In another embodiment, the invention provides vaccine preparationscomprising the CoV-HKU1 recombinant and chimeric forms of said virus, orsubunits of the virus.

The present invention further provides methods of preparing recombinantor chimeric forms of CoV-HKU1. In another specific embodiment, thevaccine preparations of the present invention comprise one or morenucleic acid molecules comprising or consisting of the sequence of SEQID NO:1 and/or 3, or a fragment thereof. In another embodiment, theinvention provides vaccine preparations comprising one or morepolypeptides of the invention encoded by a nucleotide sequencecomprising or consisting of the nucleotide sequence of SEQ ID NO:1and/or 3, or a fragment thereof, including the polypeptides having theamino acid sequences of SEQ ID NO:2 or SEQ ID NOS:34-2918 shown in FIGS.2 and 8. Furthermore, the present invention provides methods fortreating, ameliorating, managing, or preventing respiratory tractinfections by administering to a subject in need thereof the anti-viralagents of the present invention, alone or in combination with otherantivirals [e.g., amantadine, rimantadine, gancyclovir, acyclovir,ribavirin, penciclovir, oseltamivir, foscarnet zidovudine (AZT),didanosine (ddI), lamivudine (3TC), zalcitabine (ddC), stavudine (d4T),nevirapine, delavirdine, indinavir, ritonavir, vidarabine, nelfinavir,saquinavir, relenza, tamiflu, pleconaril, interferons, etc.], steroidsand corticosteroids such as prednisone, cortisone, fluticasone andglucocorticoid, antibiotics, analgesics, bronchodialaters, or othertreatments for respiratory and/or viral infections. In one aspect, theanti-viral agent of the present invention prevents or inhibit thebinding of the virus or viral proteins to a host cell under aphysiological condition, thereby preventing or inhibiting the infectionof the host cell by the virus. In another aspect, the anti-viral agentof the invention prevents or inhibits replication of the viral nucleicacid molecules in the host cell under a physiological condition byinteracting with the viral nucleic acid molecules or its transcriptionmechanisms. In a specific embodiment, the anti-viral agent of theinvention includes the vaccine or immunogenic preparations of theinvention or an antibody that immunospecifically binds CoV-HKU1 or anyCoV-HKU1 epitope and may neutralizes CoV-HKU1. In another specificembodiment, the anti-viral agent is a polypeptide or protein of theinvention or a nucleic acid molecule of the invention. In addition, thepresent invention provides a method of preventing or inhibitingreplication in a host cell of a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:1 and/or 3, or inhibiting theactivities of the polypeptides encoded by the nucleotide sequence of SEQID NO:1 and/or 3, a complement thereof, or a portion thereof, includingthe polypeptides having the amino acid sequence of SEQ ID NO:2 or SEQ IDNO:34-2918 shown in FIGS. 2 and 8, by administering to said host cellthe anti-viral agent of the invention. In a specific embodiment the hostcell is a mammalian cell, such as a cell of humans, primates, horses,cows, sheep, pigs, goats, dogs, cats, arivan species and rodents.Preferably, the cell is a primate cell and most preferably a human cell.

Furthermore, the present invention provides pharmaceutical compositionscomprising anti-viral agents of the present invention and apharmaceutically acceptable carrier. The present invention also provideskits comprising pharmaceutical compositions of the present invention.

5.1 Recombinant and Chimeric CoV-HKU1

The present invention encompasses recombinant or chimeric virusesencoded by viral vectors derived from the genome of CoV-HKU1 or naturalvariants thereof. In a specific embodiment, a recombinant virus is onederived from the CoV-HKU1. In a specific embodiment, the virus has anucleotide sequence of SEQ ID NO:3. In another specific embodiment, arecombinant virus is one derived from a natural variant of CoV-HKU1. Anatural variant of CoV-HKU1 has a sequence that is different from thegenomic sequence (SEQ ID NO:3) of CoV-HKU1, due to one or more naturallyoccurred mutations, including, but not limited to, point mutations,rearrangements, insertions, deletions etc., to the genomic sequence thatmay or may not result in a phenotypic change. In accordance with thepresent invention, a viral vector which is derived from the genome ofthe CoV-HKU, is one that contains a nucleic acid sequence that encodesat least a part of one ORF of the CoV-HKU1. In a specific embodiment,the ORF comprises or consists of a nucleotide sequence of SEQ ID NO:1 ora fragment thereof. In a specific embodiment, there are more than oneORF within the nucleotide sequence of SEQ ID NO:3, or a fragmentthereof. In another embodiment, the polypeptides encoded by the ORFcomprises or consists of amino acid sequences of SEQ ID NO:34-2918 shownin FIGS. 2 and 8, or SEQ ID NO:2, or a fragment thereof. In accordancewith the present invention these viral vectors may or may not includenucleic acids that are non-native to the viral genome.

In another specific embodiment, a chimeric virus of the invention is arecombinant CoV-HKU1 which further comprises a heterologous nucleotidesequence. In accordance with the invention, a chimeric virus may beencoded by a nucleotide sequence in which heterologous nucleotidesequences have been added to the genome or in which endogenous or nativenucleotide sequences have been replaced with heterologous nucleotidesequences.

According to the present invention, the chimeric viruses are encoded bythe viral vectors of the invention which further comprise a heterologousnucleotide sequence. In accordance with the present invention a chimericvirus is encoded by a viral vector that may or may not include nucleicacids that are non-native to the viral genome. In accordance with theinvention a chimeric virus is encoded by a viral vector to whichheterologous nucleotide sequences have been added, inserted orsubstituted for native or non-native sequences. In accordance with thepresent invention, the chimeric virus may be encoded by nucleotidesequences derived from different strains or variants of CoV-HKU1. Inparticular, the chimeric virus is encoded by nucleotide sequences thatencode antigenic polypeptides derived from different strains or variantsof CoV-HKU1.

A chimeric virus may be of particular use for the generation ofrecombinant vaccines protecting against two or more viruses (Tao et al.,J. Virol. 72, 2955-2961; Durbin et al., 2000, J. Virol. 74, 6821-6831;Skiadopoulos et al., 1998, J. Virol. 72, 1762-1768 (1998); Teng et al.,2000, J. Virol. 74, 9317-9321). For example, it can be envisaged that avirus vector derived from the CoV-HKU1 expressing one or more proteinsof variants of CoV-HKU1, or vice versa, will protect a subjectvaccinated with such vector against infections by both the nativeCoV-HKU1 and the variant. Attenuated and replication-defective virusesmay be of use for vaccination purposes with live vaccines as has beensuggested for other viruses.

In accordance with the present invention the heterologous sequence to beincorporated into the viral vectors encoding the recombinant or chimericviruses of the invention include sequences obtained or derived fromdifferent strains or variants of CoV-HKU1.

In certain embodiments, the chimeric or recombinant viruses of theinvention are encoded by viral vectors derived from viral genomeswherein one or more sequences, intergenic regions, termini sequences, orportions or entire ORF have been substituted with a heterologous ornon-native sequence. In certain embodiments of the invention, thechimeric viruses of the invention are encoded by viral vectors derivedfrom viral genomes wherein one or more heterologous sequences have beeninserted or added to the vector.

The selection of the viral vector may depend on the species of thesubject that is to be treated or protected from a viral infection.

In accordance with the present invention, the viral vectors can beengineered to provide antigenic sequences which confer protectionagainst infection by the CoV-HKU1 and natural variants thereof. Theviral vectors may be engineered to provide one, two, three or moreantigenic sequences. In accordance with the present invention theantigenic sequences may be derived from the same virus, from differentstrains or variants of the same type of virus, or from differentviruses.

The expression products and/or recombinant or chimeric virions obtainedin accordance with the invention may advantageously be utilized invaccine formulations. The expression products and chimeric virions ofthe present invention may be engineered to create vaccines against abroad range of pathogens, including viral and bacterial antigens, tumorantigens, allergen antigens, and auto antigens involved in autoimmunedisorders. In particular, the chimeric virions of the present inventionmay be engineered to create vaccines for the protection of a subjectfrom infections with CoV-HKU1 and variants thereof.

In certain embodiments, the expression products and recombinant orchimeric virions of the present invention may be engineered to createvaccines against a broad range of pathogens, including viral antigens,tumor antigens and autoantigens involved in autoimmune disorders. Oneway to achieve this goal involves modifying existing CoV-HKU1 genes tocontain foreign sequences in their respective external domains. Wherethe heterologous sequences are epitopes or antigens of pathogens, thesechimeric viruses may be used to induce a protective immune responseagainst the disease agent from which these determinants are derived.

Thus, the present invention relates to the use of viral vectors andrecombinant or chimeric viruses to formulate vaccines against a broadrange of viruses and/or antigens. The present invention also encompassesrecombinant viruses comprising a viral vector derived from the CoV-HKU1or variants thereof which contains sequences which result in a virushaving a phenotype more suitable for use in vaccine formulations. Themutations and modifications can be in coding regions, in intergenicregions and in the leader and trailer sequences of the virus.

The invention provides a host cell comprising a nucleic acid or a vectoraccording to the invention. Plasmid or viral vectors containing thepolymerase components of CoV-HKU1 are generated in prokaryotic cells forthe expression of the components in relevant cell types (bacteria,insect cells, eukaryotic cells). Plasmid or viral vectors containingfull-length or partial copies of the CoV-HKU1 genome will be generatedin prokaryotic cells for the expression of viral nucleic acids in-vitroor in-vivo. The latter vectors may contain other viral sequences for thegeneration of chimeric viruses or chimeric virus proteins, may lackparts of the viral genome for the generation of replication defectivevirus, and may contain mutations, deletions or insertions for thegeneration of attenuated viruses.

In addition, eukaryotic cells, transiently or stably expressing one ormore full-length or partial CoV-HKU1 proteins can be used. Such cellscan be made by transfection (proteins or nucleic acid vectors),infection (viral vectors) or transduction (viral vectors) and may beuseful for complementation of mentioned wild type, attenuated,replication-defective or chimeric viruses.

The viral vectors and chimeric viruses of the present invention may beused to modulate a subject's immune system by stimulating a humoralimmune response, a cellular immune response or by stimulating toleranceto an antigen. As used herein, a subject means: humans, primates,horses, cows, sheep, pigs, goats, dogs, cats, avian species and rodents.

5.2 Formulation of Vaccines and Antivirals

In a preferred embodiment, the invention provides a proteinaceousmolecule or CoV-HKU1 specific viral protein or functional fragmentthereof encoded by a nucleic acid according to the invention. Usefulproteinaceous molecules are for example derived from any of the genes orgenomic fragments derivable from the virus according to the invention,including envelop protein (E protein), integral membrane protein (Mprotein), spike protein (S protein), nucleocapsid protein (N protein),hemagglutinin esterase (HE protein), and RNA-dependent RNA polymerase.Such molecules, or antigenic fragments thereof, as provided herein, arefor example useful in diagnostic methods or kits and in pharmaceuticalcompositions such as subunit vaccines. Particularly useful arepolypeptides encoded by the nucleotide sequence of SEQ ID NO:1 or 3,including the polypeptides having the amino acid sequences of SEQ IDNOS:34-2918 in FIGS. 2 and 8, or SEQ ID NO:2, or antigenic fragmentsthereof for inclusion as antigen or subunit immunogen, but inactivatedwhole virus can also be used. Particularly useful are also thoseproteinaceous substances that are encoded by recombinant nucleic acidfragments of the CoV-HKU1 genome; of course preferred are those that arewithin the preferred bounds and metes of ORFs, in particular, foreliciting CoV-HKU1 specific antibody or T cell responses, whether invivo (e.g. for protective or therapeutic purposes or for providingdiagnostic antibodies) or in vitro (e.g. by phage display technology oranother technique useful for generating synthetic antibodies).

The invention provides vaccine formulations for the prevention andtreatment of infections with CoV-HKU1. In certain embodiments, thevaccine of the invention comprises recombinant and chimeric viruses ofthe CoV-HKU1.

In another aspect, the present invention also provides DNA vaccineformulations comprising a nucleic acid or fragment of the CoV-HKU1, ornucleic acid molecules having the sequence of SEQ ID NO:1 or 3, or afragment thereof. In another specific embodiment, the DNA vaccineformulations of the present invention comprises a nucleic acid orfragment thereof encoding the antibodies which immunospecifically bindsCoV-HKU1. In DNA vaccine formulations, a vaccine DNA comprises a viralvector, such as that derived from the CoV-HKU1, bacterial plasmid, orother expression vector, bearing an insert comprising a nucleic acidmolecule of the present invention operably linked to one or more controlelements, thereby allowing expression of the vaccinating proteinsencoded by said nucleic acid molecule in a vaccinated subject. Suchvectors can be prepared by recombinant DNA technology as recombinant orchimeric viral vectors carrying a nucleic acid molecule of the presentinvention.

Various heterologous vectors are described for DNA vaccinations againstviral infections. For example, the vectors described in the followingreferences may be used to express CoV-HKU1 sequences instead of thesequences of the viruses or other pathogens described; in particular,vectors described for hepatitis B virus (Michel, M. L. et al., 1995,DAN-mediated immunization to the hepatitis B surface antigen in mice:Aspects of the humoral response mimic hepatitis B viral infection inhumans, Proc. Natl. Aca. Sci. USA 92:5307-5311; Davis, H. L. et al.,1993, DNA-based immunization induces continuous seretion of hepatitis Bsurface antigen and high levels of circulating antibody, Human Molec.Genetics 2:1847-1851), HIV virus (Wang, B. et al., 1993, Geneinoculation generates immune responses against human immunodeficiencyvirus type 1, Proc. Natl. Acad. Sci. USA 90:4156-4160; Lu, S. et al.,1996, Simian immunodeficiency virus DNA vaccine trial in macques, J.Virol. 70:3978-3991; Letvin, N. L. et al., 1997, Potent, protectiveanti-HIV immune responses generated by bimodal HIV envelope DNA plusprotein vaccination, Proc Natl Acad Sci USA. 94(17):9378-83), andinfluenza viruses (Robinson, H L et al., 1993, Protection against alethal influenza virus challenge by immunization with ahaemagglutinin-expressing plasmid DNA, Vaccine 11:957-960; Ulmer, J. B.et al., Heterologous protection against influenza by injection of DNAencoding a viral protein, Science 259:1745-1749), as well as bacterialinfections, such as tuberculosis (Tascon, R. E. et al., 1996,Vaccination against tuberculosis by DNA injection, Nature Med.2:888-892; Huygen, K. et al., 1996, Immunogenicity and protectiveefficacy of a tuberculosis DNA vaccine, Nature Med., 2:893-898), andparasitic infection, such as malaria (Sedegah, M., 1994, Protectionagainst malaria by immunization with plasmid DNA encodingcircumsporozoite protein, Proc. Natl. Acad. Sci. USA 91:9866-9870;Doolan, D. L. et al., 1996, Circumventing genetic restriction ofprotection against malaria with multigene DNA immunization: CD8+ Tcell-interferon δ, and nitric oxide-dependent immunity, J. Exper. Med.,1183:1739-1746).

Many methods may be used to introduce the vaccine formulations describedabove. These include, but are not limited to, oral, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, andintranasal routes. Alternatively, it may be preferable to introduce thechimeric virus vaccine formulation via the natural route of infection ofthe pathogen for which the vaccine is designed. The DNA vaccines of thepresent invention may be administered in saline solutions by injectionsinto muscle or skin using a syringe and needle (Wolff J. A. et al.,1990, Direct gene transfer into mouse muscle in vivo, Science247:1465-1468; Raz, E., 1994, Intradermal gene immunization: Thepossible role of DNA uptake in the induction of cellular immunity toviruses, Proc. Natl. Acd. Sci. USA 91:9519-9523). Another way toadminister DNA vaccines is called “gene gun” method, whereby microscopicgold beads coated with the DNA molecules of interest is fired into thecells (Tang, D. et al., 1992, Genetic immunization is a simple methodfor eliciting an immune response, Nature 356:152-154). For generalreviews of the methods for DNA vaccines, see Robinson, H. L., 1999, DNAvaccines: basic mechanism and immune responses (Review), Int. J. Mol.Med. 4(5):549-555; Barber, B., 1997, Introduction: Emerging vaccinestrategies, Seminars in Immunology 9(5):269-270; and Robinson, H. L. etal., 1997, DNA vaccines, Seminars in Immunology 9(5):271-283.

5.3 Adjuvants and Carrier Molecules

CoV-HKU1-associated antigens are administered with one or moreadjuvants. In one embodiment, the CoV-HKU1-associated antigen isadministered together with a mineral salt adjuvants or mineral salt geladjuvant. Such mineral salt and mineral salt gel adjuvants include, butare not limited to, aluminum hydroxide (ALHYDROGEL, REHYDRAGEL),aluminum phosphate gel, aluminum hydroxyphosphate (ADJU-PHOS), andcalcium phosphate.

In another embodiment, CoV-HKU1-associated antigen is administered withan immunostimulatory adjuvant. Such class of adjuvants, include, but arenot limited to, cytokines (e.g., interleukin-2, interleukin-7,interleukin-12, granulocyte-macrophage colony stimulating factor(GM-CSF), interfereon-γ interleukin-1β (IL-1β), and IL-1β peptide orSclavo Peptide), cytokine-containing liposomes, triterpenoid glycosidesor saponins (e.g., QuilA and QS-21, also sold under the trademarkSTIMULON, ISCOPREP), Muramyl Dipeptide (MDP) derivatives, such asN-acetyl-muramyl-L-threonyl-D-isoglutamine (Threonyl-MDP, sold under thetrademark TERMURTIDE), GMDP,N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine,N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine, muramyl tripeptide phosphatidylethanolamine(MTP-PE), unmethylated CpG dinucleotides and oligonucleotides, such asbacterial DNA and fragments thereof, LPS, monophosphoryl Lipid A (3D-MLAsold under the trademark MPL), and polyphosphazenes.

In another embodiment, the adjuvant used is a particular adjuvant,including, but not limited to, emulsions, e.g., Freund's CompleteAdjuvant, Freund's Incomplete Adjuvant, squalene or squalaneoil-in-water adjuvant formulations, such as SAF and MF59, e.g., preparedwith block-copolymers, such as L-121 (polyoxypropylene/polyoxyetheylene)sold under the trademark PLURONIC L-121, Liposomes, Virosomes,cochleates, and immune stimulating complex, which is sold under thetrademark ISCOM.

In another embodiment, a microparticular adjuvant is used.Microparticulare adjuvants include, but are not limited to biodegradableand biocompatible polyesters, homo- and copolymers of lactic acid (PLA)and glycolic acid (PGA), poly(lactide-co-glycolides) (PLGA)microparticles, polymers that self-associate into particulates(poloxamer particles), soluble polymers (polyphosphazenes), andvirus-like particles (VLPs) such as recombinant protein particulates,e.g., hepatitis B surface antigen (HbsAg).

Yet another class of adjuvants that may be used include mucosaladjuvants, including but not limited to heat-labile enterotoxin fromEscherichia coli (LT), cholera holotoxin (CT) and cholera Toxin BSubunit (CTB) from Vibrio cholerae, mutant toxins (e.g., LTK63 andLTR72), microparticles, and polymerized liposomes.

In other embodiments, any of the above classes of adjuvants may be usedin combination with each other or with other adjuvants. For example,non-limiting examples of combination adjuvant preparations that can beused to administer the CoV-HKU1-associated antigens of the inventioninclude liposomes containing immunostimulatory protein, cytokines, orT-cell and/or B-cell peptides, or microbes with or without entrappedIL-2 or microparticles containing enterotoxin. Other adjuvants known inthe art are also included within the scope of the invention (see VaccineDesign: The Subunit and Adjuvant Approach, Chap. 7, Michael F. Powelland Mark J. Newman (eds.), Plenum Press, New York, 1995, which isincorporated herein in its entirety).

The effectiveness of an adjuvant may be determined by measuring theinduction of antibodies directed against an immunogenic polypeptidecontaining a CoV-HKU1 polypeptide epitope, the antibodies resulting fromadministration of this polypeptide in vaccines which are also comprisedof the various adjuvants.

The polypeptides may be formulated into the vaccine as neutral or saltforms. Pharmaceutically acceptable salts include the acid additionalsalts (formed with free amino groups of the peptide) and which areformed with inorganic acids, such as, for example, hydrochloric orphosphoric acids, or organic acids such as acetic, oxalic, tartaric,maleic, and the like. Salts formed with free carboxyl groups may also bederived from inorganic bases, such as, for example, sodium potassium,ammonium, calcium, or ferric hydroxides, and such organic bases asisopropylamine, trimethylamine, 2-ethylamino ethanol, histidine,procaine and the like.

The vaccines of the invention may be multivalent or univalent.Multivalent vaccines are made from recombinant viruses that direct theexpression of more than one antigen.

Many methods may be used to introduce the vaccine formulations of theinvention; these include but are not limited to oral, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasalroutes, and via scarification (scratching through the top layers ofskin, e.g., using a bifurcated needle).

The patient to which the vaccine is administered is preferably a mammal,most preferably a human, but can also be a non-human animal includingbut not limited to cows, horses, sheep, pigs, fowl (e.g., chickens),goats, cats, dogs, hamsters, mice and rats.

5.4 Preparation of Antibodies

Antibodies which specifically recognize a polypeptide of the invention,such as, but not limited to, polypeptides comprising the sequence of SEQID NO:2 or any of SEQ ID NOS: 34-2918 or CoV-HKU1 epitope, orantigen-binding fragments thereof, can be used for detecting, screening,and isolating the polypeptide of the invention or fragments thereof, orsimilar sequences that might encode similar enzymes from the otherorganisms. For example, in one specific embodiment, an antibody whichimmunospecifically binds CoV-HKU1 epitope, or a fragment thereof, can beused for various in vitro detection assays, including enzyme-linkedimmunosorbent assays (ELISA), radioimmunoassays, Western blot, etc., forthe detection of a polypeptide of the invention or, preferably,CoV-HKU1, in samples, for example, a biological material, includingcells, cell culture media (e.g., bacterial cell culture media, mammaliancell culture media, insect cell culture media, yeast cell culture media,etc.), blood, plasma, serum, tissues, sputum, naseopharyngeal aspirates,etc.

Antibodies specific for a polypeptide of the invention or any epitope ofCoV-HKU1 may be generated by any suitable method known in the art.Polyclonal antibodies to an antigen-of-interest, for example, theCoV-HKU1 epitopes or polypeptides encoded by a nucleotide sequence ofSEQ ID NO:1 or 3, including the polypeptides shown in FIG. 2 (SEQ IDNOS:34-1318), FIG. 8 (SEQ ID NOS:1319-2918), as well as SEQ ID NO:2, canbe produced by various procedures well known in the art. For example, anantigen can be administered to various host animals including, but notlimited to, rabbits, mice, rats, etc., to induce the production ofantisera containing polyclonal antibodies specific for the antigen.Various adjuvants may be used to increase the immunological response,depending on the host species, and include but are not limited to,Freund's (complete and incomplete) adjuvant, mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful adjuvants for humanssuch as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum. Suchadjuvants are also well known in the art (see Section 5.4, supra).

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas, pp. 563-681 (Elsevier, N.Y., 1981) (both of which areincorporated by reference in their entireties). The term “monoclonalantibody” as used herein is not limited to antibodies produced throughhybridoma technology. The term “monoclonal antibody” refers to anantibody that is derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. In anon-limiting example, mice can be immunized with an antigen of interestor a cell expressing such an antigen. Once an immune response isdetected, e.g., antibodies specific for the antigen are detected in themouse serum, the mouse spleen is harvested and splenocytes isolated. Thesplenocytes are then fused by well known techniques to any suitablemyeloma cells. Hybridomas are selected and cloned by limiting dilution.The hybridoma clones are then assayed by methods known in the art forcells that secrete antibodies capable of binding the antigen. Ascitesfluid, which generally contains high levels of antibodies, can begenerated by inoculating mice intraperitoneally with positive hybridomaclones.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)₂ fragments may be producedby proteolytic cleavage of immunoglobulin molecules, using enzymes suchas papain (to produce Fab fragments) or pepsin (to produce F(ab′)₂fragments). F(ab′)₂ fragments contain the complete light chain, and thevariable region, the CH1 region and the hinge region of the heavy chain.

The antibodies of the invention or fragments thereof can be alsoproduced by any method known in the art for the synthesis of antibodies,in particular, by chemical synthesis or preferably, by recombinantexpression techniques.

The nucleotide sequence encoding an antibody may be obtained from anyinformation available to those skilled in the art (i.e., from Genbank,the literature, or by routine cloning and sequence analysis). If a clonecontaining a nucleic acid encoding a particular antibody or anepitope-binding fragment thereof is not available, but the sequence ofthe antibody molecule or epitope-binding fragment thereof is known, anucleic acid encoding the immunoglobulin may be chemically synthesizedor obtained from a suitable source (e.g., an antibody cDNA library, or acDNA library generated from, or nucleic acid, preferably poly A+ RNA,isolated from any tissue or cells expressing the antibody, such ashybridoma cells selected to express an antibody) by PCR amplificationusing synthetic primers hybridizable to the 3′ and 5′ ends of thesequence or by cloning using an oligonucleotide probe specific for theparticular gene sequence to identify, e.g., a cDNA clone from a cDNAlibrary that encodes the antibody. Amplified nucleic acids generated byPCR may then be cloned into replicable cloning vectors using any methodwell known in the art.

Once the nucleotide sequence of the antibody is determined, thenucleotide sequence of the antibody may be manipulated using methodswell known in the art for the manipulation of nucleotide sequences,e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc.(see, for example, the techniques described in Sambrook et al., supra;and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology,John Wiley & Sons, NY, which are both incorporated by reference hereinin their entireties), to generate antibodies having a different aminoacid sequence by, for example, introducing amino acid substitutions,deletions, and/or insertions into the epitope-binding domain regions ofthe antibodies or any portion of antibodies which may enhance or reducebiological activities of the antibodies.

Recombinant expression of an antibody requires construction of anexpression vector containing a nucleotide sequence that encodes theantibody. Once a nucleotide sequence encoding an antibody molecule or aheavy or light chain of an antibody, or portion thereof has beenobtained, the vector for the production of the antibody molecule may beproduced by recombinant DNA technology using techniques well known inthe art as discussed in the previous sections. Methods which are wellknown to those skilled in the art can be used to construct expressionvectors containing antibody coding sequences and appropriatetranscriptional and translational control signals. These methodsinclude, for example, in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. The nucleotide sequenceencoding the heavy-chain variable region, light-chain variable region,both the heavy-chain and light-chain variable regions, anepitope-binding fragment of the heavy- and/or light-chain variableregion, or one or more complementarity determining regions (CDRs) of anantibody may be cloned into such a vector for expression. Thus-preparedexpression vector can be then introduced into appropriate host cells forthe expression of the antibody. Accordingly, the invention includes hostcells containing a polynucleotide encoding an antibody specific for thepolypeptides of the invention or fragments thereof.

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides or different selectablemarkers to ensure maintenance of both plasmids. Alternatively, a singlevector may be used which encodes, and is capable of expressing, bothheavy and light chain polypeptides. In such situations, the light chainshould be placed before the heavy chain to avoid an excess of toxic freeheavy chain (Proudfoot, Nature, 322:52, 1986; and Kohler, Proc. Natl.Acad. Sci. USA, 77:2 197, 1980). The coding sequences for the heavy andlight chains may comprise cDNA or genomic DNA.

In another embodiment, antibodies can also be generated using variousphage display methods known in the art. In phage display methods,functional antibody domains are displayed on the surface of phageparticles which carry the polynucleotide sequences encoding them. In aparticular embodiment, such phage can be utilized to display antigenbinding domains, such as Fab and Fv or disulfide-bond stabilized Fv,expressed from a repertoire or combinatorial antibody library (e.g.,human or murine). Phage expressing an antigen binding domain that bindsthe antigen of interest can be selected or identified with antigen,e.g., using labeled antigen or antigen bound or captured to a solidsurface or bead. Phage used in these methods are typically filamentousphage, including fd and M13. The antigen binding domains are expressedas a recombinantly fused protein to either the phage gene III or geneVIII protein. Examples of phage display methods that can be used to makethe immunoglobulins, or fragments thereof, of the present inventioninclude those disclosed in Brinkman et al., J. Immunol. Methods,182:41-50, 1995; Ames et al., J. Immunol. Methods, 184:177-186, 1995;Kettleborough et al., Eur. J. Immunol., 24:952-958, 1994; Persic et al.,Gene, 187:9-18, 1997; Burton et al., Advances in Immunology, 57:191-280,1994; PCT application No. PCT/GB91/01134; PCT publications WO 90/02809;WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717;5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637;5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which isincorporated herein by reference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired fragments, and expressed in any desired host, includingmammalian cells, insect cells, plant cells, yeast, and bacteria, e.g.,as described in detail below. For example, techniques to recombinantlyproduce Fab, Fab′ and F(ab)₂ fragments can also be employed usingmethods known in the art such as those disclosed in PCT publication WO92/22324; Mullinax et al., BioTechniques, 12(6):864-869, 1992; and Sawaiet al., AJRI, 34:26-34, 1995; and Better et al., Science, 240:1041-1043,1988 (each of which is incorporated by reference in its entirety).Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., Methods in Enzymology, 203:46-88, 1991; Shu etal., PNAS, 90:7995-7999, 1993; and Skerra et al., Science,240:1038-1040, 1988.

Once an antibody molecule of the invention has been produced by anymethods described above, it may then be purified by any method known inthe art for purification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A or Protein G purification, andsizing column chromatography), centrifugation, differential solubility,or by any other standard techniques for the purification of proteins.Further, the antibodies of the present invention or fragments thereofmay be fused to heterologous polypeptide sequences described herein orotherwise known in the art to facilitate purification.

For some uses, including in vivo use of antibodies in humans and invitro detection assays, it may be preferable to use chimeric, humanized,or human antibodies. A chimeric antibody is a molecule in whichdifferent portions of the antibody are derived from different animalspecies, such as antibodies having a variable region derived from amurine monoclonal antibody and a constant region derived from a humanimmunoglobulin. Methods for producing chimeric antibodies are known inthe art. See e.g., Morrison, Science, 229:1202, 1985; Oi et al.,BioTechniques, 4:214 1986; Gillies et al., J. Immunol. Methods,125:191-202, 1989; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397,which are incorporated herein by reference in their entireties.Humanized antibodies are antibody molecules from non-human species thatbind the desired antigen having one or more complementarity determiningregions (CDRs) from the non-human species and framework regions from ahuman immunoglobulin molecule. Often, framework residues in the humanframework regions will be substituted with the corresponding residuefrom the CDR donor antibody to alter, preferably improve, antigenbinding. These framework substitutions are identified by methods wellknown in the art, e.g., by modeling of the interactions of the CDR andframework residues to identify framework residues important for antigenbinding and sequence comparison to identify unusual framework residuesat particular positions. See, e.g., Queen et al., U.S. Pat. No.5,585,089; Riechmann et al., Nature, 332:323, 1988, which areincorporated herein by reference in their entireties. Antibodies can behumanized using a variety of techniques known in the art including, forexample, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101 and 5,585,089), veneering or resurfacing(EP 592,106; EP 519,596; Padlan, Molecular Immunology, 28(4/5):489-498,1991; Studnicka et al., Protein Engineering, 7(6):805-814, 1994; Roguskaet al., Proc Natl. Acad. Sci. USA, 91:969-973, 1994), and chainshuffling (U.S. Pat. No. 5,565,332), all of which are herebyincorporated by reference in their entireties.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645; WO 98/50433; WO 98/24893; WO 98/16654; WO96/34096; WO 96/33735; and WO 91/10741, each of which is incorporatedherein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For an overview of thistechnology for producing human antibodies, see Lonberg and Huszar, Int.Rev. Immunol., 13:65-93, 1995. For a detailed discussion of thistechnology for producing human antibodies and human monoclonalantibodies and protocols for producing such antibodies, see, e.g., PCTpublications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735;European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923; 5,625,126;5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793;5,916,771; and 5,939,598, which are incorporated by reference herein intheir entireties. In addition, companies such as Abgenix, Inc. (Fremont,Calif.), Medarex (NJ) and Genpharm (San Jose, Calif.) can be engaged toprovide human antibodies directed against a selected antigen usingtechnology similar to that described above.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology,12:899-903, 1988).

Antibodies fused or conjugated to heterologous polypeptides may be usedin in vitro immunoassays and in purification methods (e.g., affinitychromatography) well known in the art. See e.g., PCT publication NumberWO 93/21232; EP 439,095; Naramura et al., Immunol. Lett., 39:91-99,1994; U.S. Pat. No. 5,474,981; Gillies et al., PNAS, 89:1428-1432, 1992;and Fell et al., J. Immunol., 146:2446-2452, 1991, which areincorporated herein by reference in their entireties.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the polypeptidesof the invention or fragments, derivatives, analogs, or variantsthereof, or similar molecules having the similar enzymatic activities asthe polypeptide of the invention. Such solid supports include, but arenot limited to, glass, cellulose, polyacrylamide, nylon, polystyrene,polyvinyl chloride or polypropylene.

5.5 Pharmaceutical Compositions and Kits

The present invention encompasses pharmaceutical compositions comprisinganti-viral agents of the present invention. In a specific embodiment,the anti-viral agent is an antibody which immunospecifically bindsCoV-HKU1 or variants thereof, or any proteins derived therefrom. Inanother specific embodiment, the anti-viral agent is a polypeptide ornucleic acid molecule of the invention. The pharmaceutical compositionshave utility as an anti-viral prophylactic agent and may be administeredto a subject where the subject has been exposed or is expected to beexposed to a virus.

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu and Wu, 1987, J. Biol. Chem. 262:4429 4432). Methods of introductioninclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds may be administered by any convenient route,for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local. Ina preferred embodiment, it may be desirable to introduce thepharmaceutical compositions of the invention into the lungs by anysuitable route. Pulmonary administration can also be employed, e.g., byuse of an inhaler or nebulizer, and formulation with an aerosolizingagent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved by, for example, and not by way oflimitation, local infusion during surgery, topical application, e.g., inconjunction with a wound dressing after surgery, by injection, by meansof a catheter, by means of a suppository, by means of nasal spray, or bymeans of an implant, said implant being of a porous, non porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. In one embodiment, administration can be by direct injectionat the site (or former site) infected tissues.

In another embodiment, the pharmaceutical composition can be deliveredin a vesicle, in particular a liposome (see Langer, 1990, Science249:1527-1533; Treat et al., in Liposomes in the Therapy of InfectiousDisease and Cancer, Lopez Berestein and Fidler (eds.), Liss, New York,pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid.).

In yet another embodiment, the pharmaceutical composition can bedelivered in a controlled release system. In one embodiment, a pump maybe used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng.14:201; Buchwald et al., 1980, Surgery 88:507; and Saudek et al., 1989,N. Engl. J. Med. 321:574). In another embodiment, polymeric materialscan be used (see Medical Applications of Controlled Release, Langer andWise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci.Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., 1985, Science228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989,J. Neurosurg. 71:105). In yet another embodiment, a controlled releasesystem can be placed in proximity of the composition's target, i.e., thelung, thus requiring only a fraction of the systemic dose (see, e.g.,Goodson, in Medical Applications of Controlled Release, supra, vol. 2,pp. 115-138 (1984)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

The pharmaceutical compositions of the present invention comprise atherapeutically effective amount of recombinant or chimeric CoV-HKU1,and a pharmaceutically acceptable carrier. In a specific embodiment, theterm “pharmaceutically acceptable” means approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the pharmaceuticalcomposition is administered. Such pharmaceutical carriers can be sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. Water is a preferred carrier when thepharmaceutical composition is administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The composition, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. These compositions can take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained release formulations and the like. The composition can beformulated as a suppository, with traditional binders and carriers suchas triglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. The formulation should suitthe mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The pharmaceutical compositions of the invention can be formulated asneutral or salt forms. Pharmaceutically acceptable salts include thoseformed with free amino groups such as those derived from hydrochloric,phosphoric, acetic, oxalic, tartaric acids, etc., and those formed withfree carboxyl groups such as those derived from sodium, potassium,ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2ethylamino ethanol, histidine, procaine, etc.

The amount of the pharmaceutical composition of the invention which willbe effective in the treatment of a particular disorder or condition willdepend on the nature of the disorder or condition, and can be determinedby standard clinical techniques. In addition, in vitro assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances. However, suitable dosage ranges forintravenous administration are generally about 20-500 micrograms ofactive compound per kilogram body weight. Suitable dosage ranges forintranasal administration are generally about 0.01 pg/kg body weight to1 mg/kg body weight. Effective doses may be extrapolated from doseresponse curves derived from in vitro or animal model test systems.

Suppositories generally contain active ingredient in the range of 0.5%to 10% by weight; oral formulations preferably contain 10% to 95% activeingredient.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration. In apreferred embodiment, the kit contains an anti-viral agent of theinvention, e.g., an antibody specific for the polypeptides encoded by anucleotide sequence of SEQ ID NO:1 or 3, or any CoV-HKU1 epitope, or apolypeptide or protein of the present invention, including those shownin FIG. 2 (SEQ ID NOS:34-1318), FIG. 8 (SEQ ID NOS:1319-2918), and SEQID NO:2, or a nucleic acid molecule of the invention, alone or incombination with adjuvants, antivirals, antibiotics, analgesic,bronchodialaters, or other pharmaceutically acceptable excipients.

The present invention further encompasses kits comprising a containercontaining a pharmaceutical composition of the present invention andinstructions for use.

5.6 Detection Assays

The present invention provides a method for detecting an antibody, whichimmunospecifically binds to the CoV-HKU1, in a biological sample, forexample blood, serum, plasma, saliva, urine, etc., from a patientsuffering from respiratory tract infection. In a specific embodiment,the method comprising contacting the sample with the polypeptides orprotein encoded by the nucleotide sequence of SEQ ID NO:1 and/or 3,including the polypeptides having the amino acid sequences of SEQ IDNOS:34-1318 shown in FIG. 2, SEQ ID NOS:1319-2918 shown in FIG. 8, orSEQ ID NO:2, directly immobilized on a substrate and detecting thevirus-bound antibody directly or indirectly by a labeled heterologousanti-isotype antibody. In another specific embodiment, the sample iscontacted with a host cell comprising a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:1 or 3 and expressing the polypeptidesencoded thereby, and the bound antibody can be detected byimmunofluorescent assay.

An exemplary method for detecting the presence or absence of apolypeptide or nucleic acid of the invention in a biological sampleinvolves obtaining a biological sample from various sources andcontacting the sample with a compound or an agent capable of detectingan epitope or nucleic acid (e.g., mRNA, genomic RNA) of CoV-HKU1 suchthat the presence of CoV-HKU1 is detected in the sample. A preferredagent for detecting CoV-HKU1 mRNA or genomic RNA of the invention is alabeled nucleic acid probe capable of hybridizing to mRNA or genomic RNAencoding a polypeptide of the invention. The nucleic acid probe can be,for example, a nucleic acid molecule comprising or consisting of thenucleotide sequence of SEQ ID NO:1 or 3, or a portion thereof, or acomplement thereof, such as an oligonucleotide of at least 15, 20, 25,30, 50, 100, 250, 500, 750, 1,000 or more contiguous nucleotides inlength and sufficient to specifically hybridize under stringentconditions to a CoV-HKU1 mRNA or genomic RNA.

In another preferred specific embodiment, the presence of CoV-HKU1 isdetected in the sample by an reverse transcription polymerase chainreaction (RT-PCR) using the primers that are constructed based on apartial nucleotide sequence of the genome of CoV-HKU1 or a genomicnucleic acid sequence of SEQ ID NO:3, or based on a nucleotide sequenceof SEQ ID NO:1. In a non-limiting specific embodiment, preferred primersto be used in a RT-PCR method are: 5′-GGTTGGGACTATCCTAAGTGTGA-3′ (SEQ IDNO:4) and 5′-CCATCATCAGATAGAATCATCATA-3′ (SEQ ID NO:5), in the presenceof 3 mM MgCl₂ and the thermal cycles are, for example, but not limitedto, 94° C. for 8 min followed by 40 cycles of 94° C. for 1 min, 50° C.for 1 min, 72° C. for 1 min. In more preferred specific embodiment, thepresent invention provides a real-time quantitative PCR assay to detectthe presence of CoV-HKU1 in a biological sample by subjecting the cDNAobtained by reverse transcription of the extracted total RNA from thesample to PCR reactions using the specific primers, such as those havingnucleotide sequences of SEQ ID NOS:4 and 5, and a fluorescence dye, suchas SYBR® Green I, which fluoresces when bound non-specifically todouble-stranded DNA. The fluorescence signals from these reactions arecaptured at the end of extension steps as PCR product is generated overa range of the thermal cycles, thereby allowing the quantitativedetermination of the viral load in the sample based on an amplificationplot.

A preferred agent for detecting CoV-HKU1 is an antibody thatspecifically binds a polypeptide of the invention or any CoV-HKU1epitope, preferably an antibody with a detectable label. Antibodies canbe polyclonal, or more preferably, monoclonal. An intact antibody, or afragment thereof (e.g., Fab or F(ab′)₂) can be used.

The term “labeled”, with regard to the probe or antibody, is intended toencompass direct labeling of the probe or antibody by coupling (i.e.,physically linking) a detectable substance to the probe or antibody, aswell as indirect labeling of the probe or antibody by reactivity withanother reagent that is directly labeled. Examples of indirect labelinginclude detection of a primary antibody using a fluorescently labeledsecondary antibody and end-labeling of a DNA probe with biotin such thatit can be detected with fluorescently labeled streptavidin. Thedetection method of the invention can be used to detect mRNA, protein(or any epitope), or genomic RNA in a sample in vitro as well as invivo. For example, in vitro techniques for detection of mRNA includenorthern hybridizations, in situ hybridizations, RT-PCR, and RNaseprotection. In vitro techniques for detection of an epitope of CoV-HKU1include enzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence. In vitro techniques fordetection of genomic RNA include northern hybridizations, RT-PCR, andRNase protection. Furthermore, in vivo techniques for detection ofCoV-HKU1 include introducing into a subject organism a labeled antibodydirected against the polypeptide. For example, the antibody can belabeled with a radioactive marker whose presence and location in thesubject organism can be detected by standard imaging techniques,including autoradiography.

In a specific embodiment, the methods further involve obtaining acontrol sample from a control subject, contacting the control samplewith a compound or agent capable of detecting CoV-HKU1, e.g., apolypeptide of the invention or mRNA or genomic RNA encoding apolypeptide of the invention, such that the presence of CoV-HKU1 or thepolypeptide or mRNA or genomic RNA encoding the polypeptide is detectedin the sample, and comparing the absence of CoV-HKU1 or the polypeptideor mRNA or genomic RNA encoding the polypeptide in the control samplewith the presence of CoV-HKU1, or the polypeptide or mRNA or genomic DNAencoding the polypeptide in the test sample.

The invention also encompasses kits for detecting the presence ofCoV-HKU1 or a polypeptide or nucleic acid of the invention in a testsample. The kit, for example, can comprise a labeled compound or agentcapable of detecting CoV-HKU1 or the polypeptide or a nucleic acidmolecule encoding the polypeptide in a test sample and, in certainembodiments, a means for determining the amount of the polypeptide ormRNA in the sample (e.g., an antibody which binds the polypeptide or anoligonucleotide probe which binds to DNA or mRNA encoding thepolypeptide). Kits can also include instructions for use.

For antibody-based kits, the kit can comprise, for example: (1) a firstantibody (e.g., attached to a solid support) which binds to apolypeptide of the invention or CoV-HKU1 epitope; and, optionally, (2) asecond, different antibody which binds to either the polypeptide or thefirst antibody and is conjugated to a detectable agent.

For oligonucleotide-based kits, the kit can comprise, for example: (1)an oligonucleotide, e.g., a detectably labeled oligonucleotide, whichhybridizes to a nucleic acid sequence encoding a polypeptide of theinvention or to a sequence within the CoV-HKU1 genome or (2) a pair ofprimers useful for amplifying a nucleic acid molecule containing anCoV-HKU1 sequence. The kit can also comprise, e.g., a buffering agent, apreservative, or a protein stabilizing agent. The kit can also comprisecomponents necessary for detecting the detectable agent (e.g., an enzymeor a substrate). The kit can also contain a control sample or a seriesof control samples which can be assayed and compared to the test samplecontained. Each component of the kit is usually enclosed within anindividual container and all of the various containers are within asingle package along with instructions for use.

6. EXAMPLES

The following examples illustrate the identification of the novelCoV-HKU1. These examples should not be construed as limiting.

Methods and Results

As a general reference, Wiedbrauk D L & Johnston S L G. (Manual ofClinical Virology, Raven Press, New York, 1993) was used.

6.1 Clinical Subject

The patient is an in-patient of the United Christian Hospital in HongKong. Nasopharyngeal aspirates were collected from the patient weeklyfrom the first till the fifth week of the illness, stool and urine inthe first and second week of the illness, and sera in the first, second,and fourth weeks of the illness.

6.2 Antibody Detection

To produce a fusion plasmid for protein purification, primers,5′-TTTTCCTTTT GCGGCCGCTTAAGCAACAGAGTCTTCTA-3′ (SEQ ID NO:6) and5′-CGGAATTC GATGTCTTATACTCCCGGT-3′(SEQ ID NO:7) were used to amplify thegene encoding the N protein of the CoV-HKU1 by RT-PCR. The sequencecoding for amino acid residues 1 to 441 of the N protein was amplifiedand cloned into the EcoRI and NotI sites of expression vector pET-28b(+)(Novagen, Madison, Wis., USA) in frame and downstream of the series ofsix histidine residues. The (His)₆-tagged (SEQ ID NO:27) recombinant Nprotein was expressed in E. coli and purified using the Ni²⁺-loadedHiTrap Chelating System (Amersham Pharmacia, USA) according to themanufacturer's instructions.

Western blot analysis was performed as follows: Two-hundred ng ofpurified (His)₆-tagged (SEQ ID NO:27) recombinant N protein of CoV-HKU1were loaded into each well of a sodium dodecyl sulfate (SDS)-10%polyacrylamide gel and subsequently electroblotted onto a nitrocellulosemembrane (Bio-Rad, Hercules, Calif., USA). The blot was cut into stripsand the strips were incubated separately with 1:2000 dilution of serumsamples obtained during the first, second, and fourth weeks of thepatient's illness. Serum samples of two healthy blood donors were usedas controls. Antigen-antibody interaction was detected with an ECLfluorescence system (Amersham Life Science, Buckinghamshire, UK).

Several prominent immunoreactive bands were visible for serum samplescollected during the second and fourth weeks of the patient's illness(FIG. 7, lanes 2 and 3). The sizes of the largest bands were about 53kDa, consistent with the expected size of 52.8 kDa for the full-length(His)₆-tagged (SEQ ID NO:27) N protein, whereas the other bands wereconsistent with the degradation products of the (His)₆-tagged (SEQ IDNO:27) N protein. Only very faint bands were observed for serum samplesobtained from the patient during the first week of the illness (FIG. 7,lane 1) and two healthy blood donors (FIG. 7, lanes 4 and 5).

ELISA was performed using the recombinant N protein of CoV-HKU1 preparedas described above. Each well of a Nunc immunoplate (Roskilde, Denmark)was coated with 20 ng of purified (His)₆-tagged (SEQ ID NO:27)recombinant N protein for 12 h and then blocked in phosphate-bufferedsaline with 2% bovine serum albumin. The serum samples obtained from thepatient during the first, second, and fourth weeks of the illness wereserially diluted and were added to the wells of the (His)₆-tagged (SEQID NO:27) recombinant N protein-coated plates in a total volume of 100μl per well and incubated at 37° C. for 2 h. After washing with washingbuffer five times, 100 μl per well of 1:4000 diluted horse radishperoxidase-conjugated goat anti-human IgG antibody (Zymed LaboratoriesInc., South San Francisco, Calif., USA) were added to the wells andincubated at 37° C. for 1 h. After washing with washing buffer fivetimes, 100 μl of diluted 3,3′,5,5′-tetramethylbenzidine (ZymedLaboratories Inc.) were added to each well and incubated at roomtemperature for 15 min. One hundred microliters of 0.3 M H₂SO₄ wereadded and the absorbance at 450 nm of each well was measured. Eachsample was tested in duplicate and the mean absorbance for each serumwas calculated.

Box titration was carried out with different dilutions of (His)₆-tagged(SEQ ID NO:27) recombinant N protein coating antigen and serum obtainedfrom the fourth week of the patient's illness. The results identified 20ng and 80 ng of purified (His)₆-tagged recombinant N protein per ELISAwell as the ideal amount for plate coating and 1:1000 and 1:20 as themost optimal serum dilution for IgG and IgM detection, respectively.

To establish the baseline for the tests, serum samples (diluted at1:1000 and 1:20 for IgG and IgM, respectively) from 100 healthy blooddonors were tested in the CoV-HKU1 antibody ELISA. For the 100 sera fromhealthy blood donors, the mean ELISA OD₄₅₀ values for IgG and IgMdetection were 0.178 and 0.224, with standard deviations of 0.070 and0.117. Absorbance values of 0.387 and 0.576 were selected as the cutoffvalues (that equal the sum of the mean value from the healthy controland three times the standard deviation) for IgG and IgM, respectively.Using these cutoff values, the titers for IgG of the patient's serumsamples obtained during the first, second, and fourth weeks of theillness were <1:1000, 1:2000, and 1:8000, respectively (FIG. 6), andthose for IgM were 1:20, 1:40, and 1:80, respectively (data not shown).

6.3 RT-PCR and Real Time Quantitative PCR

RT-PCR Assay

An RT-PCR was developed to detect the CoV-HKU1 sequence from NPAsamples. Total RNA from clinical samples was reverse transcribed usingrandom hexamers and cDNA was amplified using primers5′-GGTTGGGACTATCCTAAGTGTGA-3′ (SEQ ID NO:4) and5′-CCATCATCAGATAGAATCATCATA-3′ (SEQ ID NO:5), which were constructedbased on the RNA-dependent RNA polymerase-encoding sequence (SEQ IDNO:1) of the CoV-HKU1 in the presence of 2.5 mM MgCl₂ (94° C. for 8 minfollowed by 40 cycles of 94° C. for 1 min, 50° C. for 1 min, 72° C. for1 min).

The summary of a typical RT-PCR protocol is as follows:

1. RNA Extraction

RNA from 140 μl of NPA samples was extracted by QIAquick® viral RNAextraction kit and was eluted in 50 μl of elution buffer.

2. Reverse Transcription

RNA 11.5 μl 0.1 M DTT 2 μl 5× buffer 4 μl 10 mM dNTP 1 μl SuperscriptII, 200 U/μl 1 μl (Invitrogen) Random hexamers, 0.3 μg/μl 0.5 μlReaction condition 42° C., 50 min 94° C., 3 min 4° C.

3. PCR

cDNA generated by random primers was amplified in a 50 μl reaction asfollows:

cDNA 2 μl 10 mM dNTP 0.5 μl 10× buffer 5 μl 25 mM MgCl₂ 5 μl 25 μMForward primer 0.5 μl 25 μM Reverse primer 0.5 μl AmpliTaq Gold ®polymerase, 5 U/μl 0.25 μl (Applied Biosystems) Water 36.25 μl

Thermal-cycle condition: 95° C., 10 min, followed by 40 cycles of 95°C., 1 min; 50° C. 1 min; 72° C., 1 min.

4. Primer Sequences

Primers were designed based on the RNA-dependent RNA polymerase encodingsequence (SEQ ID NO:1) of the CoV-HKU1.

Forward primer: 5′-GGTTGGGACTATCCTAAGTGTGA-3′ (SEQ ID NO:4)

Reverse primer: and 5′-CCATCATCAGATAGAATCATCATA-3′ (SEQ ID NO:5)

Product size: 440 bps

Real-Time Quantitative PCR Assay

Total RNA from 140 μl of nasopharyngeal aspirate (NPA) was extracted byQIAamp® virus RNA mini kit (Qiagen) as instructed by the manufacturer.Ten μl of eluted RNA samples were reverse transcribed by 200 U ofSuperscript® II reverse transcriptase (Invitrogen) in a 20 μl reactionmixture containing 0.15 μg of random hexamers, 10 mmol/L DTT, and 0.5mmol/L dNTP, as instructed. Complementary DNA was then amplified in aSYBR® Green I fluorescence reaction (Roche, Ind.) mixtures. Briefly, 20μl reaction mixtures containing 2 μl of cDNA, 3.5 mmol/L MgCl₂, 0.25μmol/L of forward primer [5′-GGTTGGGACTATCCTAAGTGTGA-3′ (SEQ ID NO:4)]and 0.25 μmol/L reverse primer [5′-CCATCATCAGATAGAATCATCATA-3′ (SEQ IDNO:5)] were thermal-cycled by a LightCycler® (Roche) with the PCRprogram, [95° C., 10 min followed by 50 cycles of 95° C., 10 min; 57°C., 5 sec; 72° C. 9 sec]. Plasmids containing the target sequence wereused as positive controls. Fluorescence signals from these reactionswere captured at the end of extension step in each cycle. To determinethe specificity of the assay, PCR products (440 base pairs) weresubjected to a melting curve analysis at the end of the assay (65° C. to95° C., 0.1° C. per second) (data not shown).

The amount of CoV-HKU1 RNA in the nasopharyngeal aspirates was followedweekly. Quantitative RT-PCR showed that the amounts of CoV-HKU1 RNA were8.5×10⁵ and 9.6×10⁶ copies per ml in two nasopharyngeal aspiratescollected in the first week of the illness, 1.5×105 copies per ml ofNPA, respectively, at two time points collected in the second week ofthe illness, but CoV-HKU1 RNA was undetectable in the NPA collected inthe third, fourth and fifth weeks of the illness (FIG. 6). CoV-HKU1 RNAwas also undetectable in the urine and stool of the patient collected inthe first and second weeks of the illness.

Discussion

The genome of CoV-HKU1 is a 29942-nucleotide long, polyadenylated RNA.The G+C content is 32%, which is the lowest among all knowncoronaviruses with genome sequences available, with a GC skew of 0.19.Table 1 shows the comparison of genomic features of CoV-HKU1 and othercorona viruses.

TABLE 1 Genome features Coronaviruses Size (bases) G + C content GC skewGroup 1 HCoV-229E 27317 0.38 0.13 PEDV 28033 0.42 0.09 HCoV-NL63 275530.34 0.16 Group 2 CoV-HKU1 29942 0.32 0.19 HCoV-OC43 30738 0.37 0.18BcoV 31028 0.37 0.17 MHV 31357 0.42 0.14 Group 3 IBV 27608 0.38 0.14SARS-CoV 29751 0.41 0.02HCoV-229E=human coronavirus 229E; PEDV=porcine epidemic diarrhea virus;HCoV-NL63=human coronavirus NL63; HCoV-OC43=human coronavirus OC43;MHV=murine hepatitis virus; BCoV=bovine coronavirus; IBV=infectiousbronchitis virus; SARS-CoV=SARS coronavirus; GC skew=(G−C)/(G+C)

The genome organization is the same as other coronaviruses, with thecharacteristic gene order 5′-replicase, S, E, M, N-3′. Both 5′ and 3′ends contain short untranslated regions. The 5′ end of the genomeconsists of a putative 5′ leader sequence. A putative transcriptionregulatory sequences (TRS) motif, 5′-CUAAAC-3′, was found at the 3′ endof the leader sequence and precedes each translated ORF except ORF5which encodes the putative E protein. Table 2 shows the putativetranscription regulatory sequences in the genome of CoV-HKU1.

TABLE 2 Number of base SEQ upstream ID of AUG ORF TRS sequence NO. −140Leader UUAAAUCUAAACUUUUUAA 8 (127) AUG −7 Hemag- UUAAAUCUAAACUAUG 9glutinin- esterase −6 Spike UUAAAUCUAAAC AUG 10 −13 ORF4UUAAAUCUAAACUUUAUUUAUG 11 −9 Membrane CUAAAUCUAAACAUUAUG 12 −13 Nucleo-UUAAAUCUAAACUAUUAGGAUG 13 capsid −35 ORF8 UUAAAUCUAAACUAUUAGGAUG 14UCUUAUACUCCCGGUCAUUAUG

As in SDAV (Sialodacryoadenitis virus) and MHV (mouse hepatitis virus),ORF5 may share the same TRS with ORF4, suggesting that the translationof the E protein is cap-independent, possibly via an internal ribosomalentry site. The 3′ untranslated region contains a predicted pseudoknotstructure 59-119 bp downstream of N gene. This pseudoknot structure ishighly conserved among coronaviruses and plays a role in coronavirus RNAreplication.

The coding potential of the CoV-HKU1 genome is shown in FIG. 3 and Table3 and the phylogenetic analyses of the chymotrypsin-like protease(3CL^(pro)), replicase, helicase, haemagglutinin-esterase (HE), S, E, Mand N, are shown in FIGS. 4A and 4B.

TABLE 3 No. of No. of amino Candidate ORFs Start-end (base) bases acidsFrame TRS ORF 1a  206-13600 13395 4465 +2 — ORF 1b 13600-21753 8154 2717+1 — HE (ORF 2) 21773-22933 1161 386 +2 Strong S (ORF 3) 22942-270124071 1356 +1 Strong ORF 4 27051-27380 330 109 +3 Strong E (ORF 5)27373-27621 249 82 +1 None M (ORF 6) 27633-28304 672 223 +3 Strong N(ORF 7) 28320-29645 1326 441 +3 Strong ORF8 28342-28959 618 205 +1Strong

The replicase 1a ORF (bases 206-13600) and replicase 1b ORF (bases13600-21753) occupy 21.5 kb of the CoV-HKU1 genome. Similar to othercoronaviruses, a frame shift interrupts the protein-coding regions andseparates the 1a and 1b ORFs. This ORF encodes a number of putativeproteins, including papain-like protease (PLP) with two copies of thePLP domain, PLP1^(pro) and PLP2^(pro), 3CL^(pro), replicase, helicase,and other proteins of unknown functions. These proteins are produced byproteolytic cleavages of a large polyprotein (FIG. 3). The sequence ofthe resulting putative proteins is the same as that in the MHV genome.This polyprotein is synthesized by a −1 ribosomal frameshift at aconserved site (UUUAAAC) upstream of a pseudoknot structure at thejunction of ORF 1a and ORF 1b. This ribosomal frameshift would result ina polyprotein of 7182 amino acids, which has 75-77% amino acididentities with the polyprotein in other Group 2 coronaviruses and43-47% amino acid identities with the polyprotein in other non-Group 2coronaviruses. The replicase gene of CoV-HKU1, which encodes 928 aminoacids, has 87-89% amino acid identities with the replicase of otherGroup 2 coronaviruses and 54-65% amino acid identities with thereplicase of other non-Group 2 coronaviruses (Table 4 and FIG. 4A).Table 4 shows amino acid identities between the predictedchymotrypsin-like protease (3CL^(pro)), replicase (Rep), helicase (Hel),hemagglutinin-esterase (HE), spike (S), envelope (E), membrane (M), andnucleocapsid (N) proteins of CoV-HKU1 and the corresponding proteins ofother coronaviruses.

TABLE 4 Pairwise amino acid identity (%) Group Virus 3CL^(pro) Rep HelHE S E M N 1 HCoV-229E 45 54 55 — 31 26 35 28 PEDV 44 56 55 — 30 34 3737 PTGV 45 57 57 — 32 34 37 27 CCoV — — — — 31 32 36 27 HCoV-NL63 43 5454 — 30 28 32 28 2 HCoV-OC43 82 87 88 57 60 54 76 58 MHV 85 89 87 50 5855 78 60 BCoV 84 88 88 56 61 55 76 57 SDAV — — — 50 61 60 77 62 ECoV — —— 53 61 56 78 59 PHEV — — — 54 61 54 77 57 3 IBV 41 60 57 — 32 28 38 27SARS- SARS-CoV 48 65 63 — 33 27 34 31 CoVHCoV-229E=human coronavirus 229E; PEDV=porcine epidemic diarrhea virus;PTGV=porcine transmissible gastroenteritis virus; CCoV=canine entericcoronavirus; HCoV-NL63=human coronavirus NL63; HCoV-OC43=humancoronavirus OC43; MHV=murine hepatitis virus; BCoV=bovine coronavirus;SDAV=rat sialodacryoadenitis coronavirus; ECoV=equine coronavirus NC99;PHEV=porcine hemagglutinating encephalomyelitis virus; IBV=infectiousbronchitis virus; SARS-CoV=SARS coronavirus

The catalytic histidine and cysteine amino acid residues, conservedamong the 3CL^(pro) in all coronaviruses, are present in the predicted3CL^(pro) of CoV-HKU1 (amino acids His³³⁷⁵ and Cys³⁴⁷⁹ of ORF 1 a). Inthe N-terminal of the putative PLP (amino acid residues 945 to 1104 ofORF 1a), there are 14 tandem copies of a 30-base repeat, which encodeNDDEDVVTGD (SEQ ID NO:15), followed by two 30-base regions that encodeNNDEEIVTGD (SEQ ID NO:16) and NDDQIVVTGD (SEQ ID NO:17), locatedupstream to the first copy of PLP domain, PLP1^(pro). This repeat is notobserved in other coronaviruses.

ORF 2 (bases 21773-22933) encodes the predicted HE glycoprotein with 386amino acids. The HE protein of CoV-HKU1 has 50-57% amino acid identitieswith the HE proteins of other Group 2 coronaviruses (Table 4 and FIG.4A). PFAM and InterProScan analyses of the ORF show that amino acidresidues 1 to 349 of the predicted protein is a member of thehaemagglutinin esterase family (PFAM accession no.: PF03996 and INTERPROaccession no. IPR007142). This family contains membrane glycoproteinsthat are present on viral surface and are involved with the cellinfection process. It contains haemagglutinin chain 1 (HE1) andhaemagglutinin chain 2 (HE2), and forms a homotrimer with each monomerbeing formed by two chains linked by a disulphide bond. Furthermore,PFAM and InterProScan analyses of the ORF show that amino acid residues122 to 236 of the predicted protein are the haemagglutinin domain ofHE-fusion glycoprotein family (PFAM accession no.: PF02710 and INTERPROaccession no. IPR003860). HE is also present in other Group 2coronaviruses and influenza C virus. SignalP analysis reveals a signalpeptide probability of 0.738, with a cleavage site between residues 13and 14. Although TMpred and TMHMM analyses of the ORF show four andthree transmembrane domains, respectively, PHDhtm analysis of the ORFshows only one transmembrane domain at positions 354 to 376. Thisconcurs with only one transmembrane region reported in the C terminal ofthe HE of BCoV (bovine coronavirus) and puffinosis virus. PrositeScananalysis of the HE protein of CoV-HKU1 reveals eight potential N-linkedglycosylation (six NXS and two NXT) sites. These are located atpositions 83 (NYT), 110, (NGS), 145 (NVS), 168 (NYS), 193 (NFS), 286(NSS), 314 (NVS, and 328 (NFT). The putative active site for neuraminateO-acetyl-esterase activity, FGDS (SEQ ID NO:18), is located at positions31-34.

ORF 3 (bases 22942-27012) encodes the predicted S glycoprotein (PFAMaccession no. PF01601) with 1356 amino acids. The S protein of CoV-HKU1has 58-61% amino acid identities with the S proteins of other Group 2coronaviruses, but has fewer than 35% amino acid identities with the Sproteins of Group 1, Group 3, and SARS-CoV (Table 4 and FIG. 4B).InterProScan analysis predicts it as a type I membrane glycoprotein.Important features of the S protein of CoV-HKU1 are depicted in FIG. 5.PrositeScan of the S protein of CoV-HKU1 reveals 28 potential N-linkedglycosylation (12 NXS and 16 NXT) sites. SignalP analysis reveals asignal peptide probability of 0.909, with a cleavage site betweenresidues 13 and 14. By multiple alignments with the S proteins of otherGroup 2 coronaviruses, a potential cleavage site located after RRKRR(SEQ ID NO:19), between residues 760 and 761, where S will be cleavedinto S1 and S2, is identified. Immediately upstream to RRKRR (SEQ IDNO:19), there is a series of five serine residues that are not presentin any other known coronaviruses (FIG. 5). Most of the S protein(residues 15 to 1300) is exposed on the outside of the virus, with atransmembrane domain at the C terminus (TMHMM analysis of the ORF showsone transmembrane domain at positions 1301 to 1356), followed by acytoplasmic tail rich in cysteine residues. Two heptad repeats (HR),located at residues 982 to 1083 (HR1) and 1250 to 1297 (HR2), identifiedby multiple alignments with other coronaviruses, are present. In MHV, ithas been confirmed that the receptor for its S protein binding isCEACAM1, a member of the carcinoembryonic antigen (CEA) family ofglycoproteins in the immunoglobulin superfamily. Furthermore, it hasbeen shown, by site-directed mutagenesis, that three conserved regions(sites I, II, and III) and some amino acid residues (Thr⁶², Thr²¹²,Tyr²¹⁴, and Tyr²¹⁶ in MHV) in the N-terminal of the S protein areparticularly important for its receptor-binding activity. By multiplealignments with the N-terminal 330 amino acids of the S protein of MHVand other group 2 coronaviruses, it is observed that these conservedregions and amino acids are present in CoV-HKU1 (FIG. 5). This infersthat the receptor for CoV-HKU1 could be a member of the CEA family onthe surface of the cells in the respiratory tract. On the other hand,for HCoV-OC43, it has been shown in vitro that the receptor for the Sprotein is a sialic acid. However, the amino acid residues on the Sprotein of HCoV-OC43 that are important for receptor binding are notwell defined.

ORF4 (bases 27051-27380) encodes a predicted protein with 109 aminoacids. This ORF overlaps with the ORF that encodes the E protein. PFAManalysis of the ORF shows that the predicted protein is a member of thecoronavirus non-structural protein NS2 family (PFAM accession no.:PF04753). TMpred and TMHMM analysis do not reveal any transmembranehelix. This predicted protein of CoV-HKU1 has 44-51% amino acididentities with the corresponding proteins of other Group 2coronaviruses.

ORF5 (bases 27373-27621) encodes the predicted E protein with 82 aminoacids. The E protein of CoV-HKU1 has 54-60% amino acid identities withthe E proteins of other Group 2 coronaviruses, but has fewer than 35%amino acid identities with the E proteins of Group 1, Group 3, andSARS-CoV (Table 4 and FIG. 4B). PFAM and InterProScan analyses of theORF show that the predicted E protein is a member of the non-structuralprotein NS3/Small envelope protein E (NS3_envE) family (PFAM accessionno.: PF02723). SignalP analysis predicts the presence of a transmembraneanchor (probability 0.995). TMpred analysis of the ORF shows twotransmembrane domains at positions 16 to 34 and 39 to 59, and TMHMManalysis of the ORF shows two transmembrane domains at positions 10 to32 and 39 to 58, consistent with the anticipated association of the Eprotein with the viral envelope. Both programs predict that both the Nand C termini are located on the surface of the virus.

ORF6 (bases 27633-28304) encodes the predicted M protein with 223 aminoacids. The M protein of CoV-HKU1 has 76-78% amino acid identities withthe M proteins of other Group 2 coronavirus, but has fewer than 40%amino acid identities with the M proteins of Group 1, Group 3, andSARS-CoV (Table 4 and FIG. 4B). PFAM analysis of the ORF shows that thepredicted M protein is a member of the coronavirus matrix glycoprotein(Corona_M) family (PFAM accession no.: PF01635). SignalP analysispredicts the presence of a transmembrane anchor (probability 0.926).TMpred analysis of the ORF shows three transmembrane domains atpositions 21 to 42, 53 to 74, and 77 to 98. TMHMM analysis of the ORFshows three transmembrane domains at positions 20 to 39, 46 to 68, and78 to 100. The N terminal 19-20 amino acids are located on the outsideand the C terminal 123-125-amino acid hydrophilic domain on the insideof the virus.

ORF7 (bases 28320-29645) encodes the predicted N protein (PFAM accessionno.: PF00937) with 441 amino acids. The N protein of CoV-HKU1 has 57-62%amino acid identities with the N proteins of other Group 2coronaviruses, but has fewer than 40% amino acid identities with the Nproteins of Group 1, Group 3, and SARS-CoV (Table 4 and FIG. 4B).

ORF8 (bases 28342-28959) encodes a hypothetical protein (N2) of 205amino acids within the ORF that encodes the predicted N protein. PFAManalysis of the ORF shows that the predicted protein is a member of thecoronavirus nucleocapsid I protein (Corona_I) family (PFAM accessionno.: PF03187). This hypothetical N2 protein of CoV-HKU1 has 32-39% aminoacid identities with the N2 proteins of other Group 2 coronaviruses.

We report the characterization and complete genome sequence of a novelcoronavirus detected in the nasopharyngeal aspirates of patients withpneumonia. The clinical significance of the virus in the first patientwas evident by the high viral loads in the patient's nasopharyngealaspirates during the first week of his illness, which coincided with theacute symptoms developed in the patient. The viral load decreased duringthe second week of the illness and was undetectable in the third week ofthe illness. In addition, the fall in viral load was accompanied by therecovery from the illness and development of specific antibody responseto the recombinant N protein of the virus. Similar to other recentlydiscovered viruses, such as hepatitis C virus, GB virus C, transfusiontransmitted virus, and SEN virus, the present virus could not berecovered from cell cultures using the standard cell lines. This couldbe related to the inherently low recovery rate of coronaviruses. Humancoronaviruses are particularly difficult to culture in vitro. Manydecades after the recognition of HCoV-229E and HCoV-OC43, there arestill only a handful of primary virus isolates available and organculture is required for primary isolation of HCoV-OC43. In ourexperience, SARS-CoV can only be recovered from less than 20% ofpatients with serologically and RT-PCR documented SARS-CoV pneumonia.Therefore, it is not surprising that the new coronavirus CoV-HKU1 hasbeen so far proven difficult to culture in vitro. After the discovery ofCoV-HKU1 in the first patient, we conducted a preliminary study on 400nasopharyngeal aspirates that were collected last year during the SARSepidemic period. Among these 400 nasopharyngeal aspirates, CoV-HKU1 wasdetected in one specimen, with a viral load comparable to that of thefirst patient. These results have suggested that CoV-HKU1 is not onlyincidentally found in one patient, but a previously unrecognizedcoronavirus associated with pneumonia.

Genomic analysis has reveals that CoV-HKU1 is a Group 2 coronavirus. Thegenome organization of CoV-HKU1 concurs with those of othercoronaviruses, with the characteristic gene order, i.e., 5′-replicase,S, E, M, N-3′, short untranslated regions in both 5′ and 3′ ends, 5′conserved coronavirus core leader sequence, putative TRS upstream tomultiple ORFs, and conserved pseudoknot in the 3′ untranslated region.In contrast to coronaviruses of other groups, CoV-HKU1 contains certainfeatures that are characteristics of Group 2 coronaviruses, includingthe presence of HE, ORF4, and N2. Phylogenetic analysis of the3CL^(pro), replicase, helicase, S, E, M, and N proteins showed thatthese genes of CoV-HKU1 were clustered with the corresponding genes inother Group 2 coronaviruses. However, the proteins of CoV-HKU1 formeddistinct branches in the phylogenetic trees, indicating that CoV-HKU1 isa distinct member of the group, and is not very closely related to anyother known members of Group 2 coronaviruses (FIGS. 4A and 4B).

In addition to phylogenetic analysis of the putative proteins, CoV-HKU1exhibits certain features that are distinct from other Group 2coronaviruses. Compared to other Group 2 coronaviruses, there is adeletion of about 800 bps between the replicase ORF 1b and the HE ORF 2in CoV-HKU1. In other Group 2 coronaviruses, including MHV, SDAV,HCoV-OC43 and BCoV, an ORF of 798-837 bp (273-278 amino acids) ispresent between the replicase 1b ORF and the HE ORF 2. This ORF encodesa protein of the coronavirus non-structural protein NS2a family (PFAMaccession no.: PF05213). The absence of this ORF in CoV-HKU1 indicatesthat this is probably a non-essential gene of coronavirus. In additionto the deletion, the N-terminal of the putative PLP in ORF 1a contains14 tandem copies of a 30-bp repeat that codes for a highly acidicdomain. Similar repeats, with different amino acid compositions, havebeen found in the genomes of human, rat and parasites, but have not beenfound in other coronaviruses. The function of these repeats is not wellunderstood, although some authors have suggested that the repeats couldbe important antigens, and their biological role may be related to theirspecial three-dimensional structures. The vitellaria antigenic proteinof Clonorchis sinensis contains 23 tandem copies of a 30-bp repeat thatcodes for DGGAQPPKSG (SEQ ID NO:20). In the case of Plasmodiumfalciparum, it has been shown that the antigenicity of thecircumsporozoite protein is due to its repeating epitope structure. Ithas also been suggested that the tandemly repeated peptide may inducestrong humoral immune response in the infected host and thus may also beuseful in serological diagnosis. Further experiments should be performedto delineate the antigenic properties, biological role, and possibleclinical usefulness of the repeat in the PLP of CoV-HKU1.

The geographical, political, and economic location of Hong Kong makes ita unique place for the study of emerging infectious disease. Hong Kong,as the gateway of southern China, with thousands of people crossing theborder on surface and by air every day, has a high potential ofimporting and exporting infectious diseases to and from China, countriesin Southeast Asia and from the rest of the world. In 1997, the first 18human cases of avian influenza A H5N1 virus infection were reported inHong Kong. In early 2003, two cases of human infection caused by avianinfluenza A (H5N1) that was acquired in Fujian, were diagnosed in HongKong, which provided an early warning of the impending disease threatfor humans and poultry in Southeast Asia that followed in 2004. For theSARS epidemic, although both epidemiological and genomic evidencerevealed that the disease had first occurred in southern China inNovember 2002, it did not receive as much international attention untilthe disease was spread to Hong Kong and through Hong Kong to Singapore,Toronto, Vietnem, and the United States of America. As for emergingbacterial infections, 50% of the patients with gastroenteritisassociated with the recovery of Laribacter hongkongensis had recenthistory of travel to southern China. In this report, one of the patientsalso had recent history of travel to Shenzhen of China prior to thedevelopment of the respiratory illness. We speculate that he might havecontacted the virus in Shenzhen. More intensive surveillance of emerginginfectious pathogens in this locality is warranted.

7. MARKET POTENTIAL

The genome of CoV-HKU1 is completely sequenced. This allows thedevelopment of various diagnostic tests as described hereinabove. Inaddition, this virus contains genetic information which is extremelyimportant and valuable for clinical and scientific researchapplications.

8. EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain manyequivalents to the specific embodiments of the invention describedherein using no more than routine experimentation. Such equivalents areintended to be encompassed by the following claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

Citation or discussion of a reference herein shall not be construed asan admission that such is prior art to the present invention.

1. An isolated nucleic acid molecule comprising the nucleotide sequenceof SEQ ID NO:3 or the complement thereof.
 2. An isolated nucleic acidmolecule comprising a nucleotide sequence having at least 45, 100, 150,200, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950, 1,000, 1,050, 1,100, 1,150, 1,200, 2,000, 3,000, 4,000, 5,000,6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000,15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 21,000, 22,000, 23,000,24,000, 25,000, 26,000, 27,000, 28,000, or 29,000 contiguous nucleotidesof the nucleotide sequence of SEQ ID NO:3, or the complement thereof. 3.The nucleic acid molecule of claim 1 or 2, wherein the molecule is RNA.4. The nucleic acid molecule of claim 1 or 2, wherein the molecule isDNA.
 5. A vector comprising the nucleic acid molecule of claim
 4. 6. Anisolated host cell comprising the vector of claim
 5. 7. An isolated hostcell comprising the nucleic acid molecule of claim 4 operably linked toa heterologous promoter.
 8. The host cell of claim 7 being a prokaryoticcell.
 9. The host cell of claim 7 is an eukaryotic cell.
 10. The hostcell of claim 9 is a mammalian cell.
 11. A method for producing apolypeptide comprising expressing the polypeptide encoded by said DNAfrom the host cell of claim 6, and recovering the polypeptide.
 12. Amethod for producing a polypeptide comprising expressing the polypeptideencoded by said DNA from the host cell of claim 7, and recovering thepolypeptide.
 13. A method for preparing a cell or progeny thereofcapable of expressing a polypeptide comprising transfecting the cellwith the vector of claim
 5. 14. A composition comprising a nucleic acidmolecule of claim 1 or 2, and a pharmaceutically acceptable carrier.